Concatameric immunoadhesion

ABSTRACT

Disclosed are concatameric proteins comprising two soluble domains, in which the C-terminus of a soluble domain of a biologically active protein is linked to the N-terminus of an identical soluble domain or a distinct soluble domain of a biologically active protein. Also, the present invention discloses dimeric proteins formed by formation of intermolecular disulfide bonds at the hinge region of two monomeric proteins formed by linkage of a concatamer of two identical soluble extracellular regions of proteins involving immune response to an Fc fragment of an immunoglobulin molecule, their glycosylated proteins, DNA constructs encoding the monomeric proteins, recombinant expression plasmids containing the DNA construct, host cells transformed or transfected with the recombinant expression plasmids, and a method of preparing the dimeric proteins by culturing the host cells. Further, the present invention discloses pharmaceutical or diagnostic compositions comprising the dimeric protein or its glycosylated form.

TECHNICAL FIELD

[0001] The present invention relates to concatameric proteins, and morespecifically, concatamerized structure of biologically active proteindomains where C-terminal end of extracellular soluble domain ofbiologically active protein is fused to N-terminal end of the same orother extracellular soluble domain of biologically active protein, anddimerization of two concatamers by coupling to hinge region of Fcfragment of immunoglobulin, and glycosylated forms of the concatamericproteins.

BACKGROUND ART

[0002] The activity of cytokine is associated with pathologic severityof inflammatory and/or immune response to various antigenicstimulations. Many antigen specific antibodies and soluble receptorswhich could recognize cytokines are currently in use to inhibit thefunction of cytokines for the therapeutic purposes (WO 93/016184, WO96/02576, WO 96/023067, WO 1997/03682, and U.S. Pat. Nos. 5,434,131,5,656,272, 5,977,318, 6,210,661, 6,225,117). Antibodies and solublereceptors inhibit cytokine signal transduction by disturbing interactionbetween cytokines and their receptors on cell surface.

[0003] Soluble receptors used as functional inhibitors of cytokine thatfused to heavy chains of human immunoglobulins were disclosed by Caponet al. (Nature 337:5254, 1989), and thereafter many patents weredisclosed inventions related to fusion proteins of soluble receptors andimmunoglobulins (U.S. Pat. Nos. 5,521,288, 5,844,095, 6,046,310,6,090,914, 6,100,383, 6,225,448).

[0004] Generally, fusion proteins of soluble receptors andimmunoglobulins have following advantages (Capon et al., Nature337:5254, 1989)

[0005] 1. Increase in total avidity to ligand by forming bivalency viadimerization.

[0006] 2. Increase in blood half-life of proteins, that is, increase inmolecular stability

[0007] 3. Activation of effecter cells by Fc fragment of immunoglobulinheavy chain

[0008] 4. Convenience of purification by using affinity column, e.g.using protein A

[0009] Most fusion proteins of receptor extracellular domain andimmunoglobulin heavy chain are composed of heavy chain without CH1domain, which result in dimers not binding to light chains. Thisstructure is more desirable for the function of proteins and receptorsinvolving immune response. For example, TNFR(WO92/16221,WO95/34326)-immunoglobulin fusion proteins disclosed in WO94/06476 andU.S. Pat. No. 5,447,851 have been used for the inhibition ofTNF-mediated inflammation. It is well known that TNFR-immunoglobulinfusion proteins have a higher affinity than original monomeric molecules(Lesslauer et al., Eur. J. Immunol. 21:2883, 1991; Ashkenazi et al.,Proc. Natl. Acad. Sci. 88:10535, 1991; Peppe et al., J. Exp. Med.174:1483, 1991; Mohler et al., J. Immunol. 151:1548, 1993).

[0010] For the improved inhibition of TNF mediated response, one canincrease efficacy by multimerizing soluble extracellular domains ofTNFR, CD2, and CTLA-4. For example, when fusion proteins of TNFR'sextracellular domains bound with immunoglobulin heavy chain(heavy chainfusion protein) and with light chain(light chain fusion protein)respectively are coexpressed in the same cell, one can produce fusionproteins as a tetrameric form by linking heavy chain to heavy and lightchains. This tetramer showed much more increased efficacy than monomericor dimeric forms as presented by Scallon et al. (Cytokine 7:759, 1995).

[0011] However, this method had many difficulties for commercializationsuch as simultaneous expression of two different fusion genes in thesame cell line, remarkably lower production yields of multimeric form;and difficulty in purifying multimeric high molecular weight forms. Forthese reasons, immunoglobulin fusion proteins currently in use are onlyheavy chain fused form.

[0012] Therefore, there is considerable demand for the development ofmethods of producing multimeric protein therapeutics with high yield andefficient purification procedures.

DISCLOSURE OF INVENTION

[0013] The present inventors have manufactured concatameric proteins byfusing the C-terminal end of soluble domain of biologically activeprotein to the N-terminal end of soluble domain of the same or otherbiologically active protein by using DNA recombination techniques. Also,the present inventors have dimerized this concatamers by linking it tothe hinge region of Fc fragment of immunoglobulin and added moreglycosylations by using DNA mutagenesis techniques. And the presentinventors have found that concatamerized protein dimers and theirglycosylated forms show increased efficacy and stability compared toconventional monomeric fusion proteins.

[0014] Therefore, in one aspect, the present invention providesconcatameric proteins where C-terminal end of soluble domain ofbiologically active proteins is fused to N-terminal end of solubledomain of the same or other biologically active proteins.

[0015] In another aspect, the present invention provides dimericproteins formed by disulfide bond at hinge region of two monomericproteins whose concatamerized part is fused to hinge region of Fcfragment of immunoglobulin.

[0016] Also in another aspect, the present invention provides DNAconstructs that encode monomeric fusion proteins whose concatamerizeddomain is fused to hinge region of Fc fragment of immunoglobulins.

[0017] Also in another aspect, the present invention provides DNAplasmids comprising a DNA construct that encodes monomeric fusionprotein whose concatamerized part is fused to hinge region of Fcfragment of immunoglobulin.

[0018] Also in another aspect, the present invention provides host cellstransfected or transformed with recombinant DNA plasmids including a DNAconstruct that encodes monomeric fusion protein whose concatamerizedpart is fused to hinge region of Fc fragment of immunoglobulin.

[0019] Also in another aspect, the present invention provides a methodfor culturing the host cells, which were transfected or transformed withrecombinant DNA plasmids including a DNA construct that encodesmonomeric fusion protein whose concatamerized part is fused to hingeregion of Fc fragment of immunoglobulin, under culture condition forexpression of DNA constructs encoding concatameric fusion proteincoupled to hinge region of Fc fragment of immunoglobulin, andmanufacturing dimeric concatamers formed by disulfide bond at hingeregion of two monomeric concatamers described as above including theprocess of purification of the proteins described as above from cellculture.

[0020] Also in another aspect, the present invention provides a methodfor culturing the host cells, which were transfected or transformed withrecombinant DNA plasmids including a DNA construct that encodesmonomeric fusion protein whose concatamerized part of immunomudulatoryfunction is fused to hinge region of Fc fragment of immunoglobulin andis inserted with glycosylation motifs, under the best condition which issuitable for expression of DNA constructs that encode monomeric fusionprotein whose concatamerized part of immune function is fused to hingeregion of Fc fragment of immunoglobulin, and for manufacturingglycosylated dimers formed by disulfide bond at hinge region of twomonomeric proteins described as above including the process ofpurification of the glycosylated proteins described as above from cellculture.

[0021] Also in another aspect, the present invention provides DNAprimers for inserting glycosylation motif into the DNA constructs thatencode monomeric fusion proteins whose concatamerized part is fused tohinge region of Fc fragment of immunoglobulins.

[0022] Also in another aspect, the present invention provides theglycosylated dimers formed by disulfide bond at hinge region of twomonomeric proteins whose concatamerized part involving immune responseis fused to hinge region of Fc fragment of immunoglobulins.

[0023] Also in another aspect, the present invention provides thepharmaceutical compositions comprising dimers formed by disulfide bondat hinge region of two monomeric proteins whose concatamerized partinvolving immune response is fused to hinge region of Fc fragment ofimmunoglobulins in a pharmaceutically effective amount and in apharmaceutically acceptable carrier.

[0024] Also in another aspect, the present invention provides thepharmaceutical compositions comprising glycosylated dimers formed bydisulfide bond at hinge region of two monomeric proteins whoseconcatamerized part involving immune response is fused to hinge regionof Fc fragment of immunoglobulins in a pharmaceutically effective amountand in a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

[0026]FIG. 1 is a schematic view showing a process of preparing a DNAconstruct encoding a conventional simple fusion monomeric proteinthrough polymerase chain reaction (PCR);

[0027]FIG. 2 is a schematic view showing a process of preparing a DNAconstruct encoding a concatameric fusion monomeric protein according tothe present invention through PCR;

[0028]FIG. 3a shows structures of [TNFR/Fc]₂, [CD2/Fc]₂ or [CTLA4/Fc]₂fusion proteins, which are simple fusion dimeric proteins formed throughhomodimerization in cells of TNFR/Fc, CD2/Fc or CTLA4/Fc fusion proteinsas examples of conventional simple fusion monomeric proteins;

[0029]FIG. 3b shows structures of [TNFR-TNFR/Fc]₂, [CD2-CD2/Fc]₂ or[CTLA4-CTLA4/Fc]₂ fusion proteins, which are concatameric fusion dimericproteins formed through homodimerization in cells of TNFR-TNFR/Fc,CD2-CD2/Fc or CTLA4-CTLA4/Fc fusion proteins as embodiments of theconcatameric fusion dimeric protein according to the present invention;

[0030]FIG. 4a shows a structure of [TNFR1-TNFR1/Fc]₂, as an embodimentof a concatameric fusion dimeric protein according to the presentinvention;

[0031]FIG. 4b shows a structure of [TNFR2-TNFR2/Fc]₂, as anotherembodiment of the concatameric fusion dimeric protein according to thepresent invention;

[0032]FIG. 4c shows a structure of [CD2-CD2/Fc]₂, as a furtherembodiment of the concatameric fusion dimeric protein according to thepresent invention;

[0033]FIG. 4d shows a structure of [CTLA4-CTLA4/Fc]₂, as a still furtherembodiment of the concatameric fusion dimeric protein according to thepresent invention;

[0034]FIG. 5 is a diagram showing a process of constructing arecombinant expression plasmid pTR11Ig-Top10′ expressing a concatamericfusion monomeric protein TNFR1-TNFR1/Fc according to the presentinvention;

[0035]FIG. 6 is a diagram showing a process of constructing arecombinant expression plasmid pCD22Ig expressing a concatameric fusionmonomeric protein CD2-CD2/Fc according to the present invention;

[0036]FIG. 7 is a map of a recombinant expression plasmid pTR11Ig-Top10′expressing a concatameric fusion monomeric protein TNFR1-TNFR1/Fcaccording to the present invention;

[0037]FIG. 8 is a map of a recombinant expression plasmid pTR22Ig-Top10′expressing a concatameric fusion monomeric protein TNFR1-TNFR1/Fcaccording to the present invention;

[0038]FIG. 9 is a map of a recombinant expression plasmid pCD22Igexpressing a concatameric fusion monomeric protein CD2-CD2/Fc accordingto the present invention;

[0039]FIG. 10 is a map of a recombinant expression plasmid pCT44Igexpressing a concatameric fusion monomeric protein CTLA4-CTLA4/Fcaccording to the present invention;

[0040]FIG. 11 is a map of a recombinant expression plasmid pTR11Ig-MGexpressing a concatameric fusion monomeric protein mgTNFR1-TNFR1/Fccontaining four glycosylation motif peptides according to the presentinvention;

[0041]FIG. 12 is a map of a recombinant expression plasmid pTR22Ig-MGexpressing a concatameric fusion monomeric protein mgTNFR2-TNFR2/Fccontaining two glycosylation motif peptides according to the presentinvention;

[0042]FIG. 13 is a map of a recombinant expression plasmid pCD22Ig-MGexpressing a concatameric fusion monomeric protein mgCD2-CD2/Fccontaining two glycosylation motif peptides according to the presentinvention;

[0043]FIG. 14 is a map of a recombinant expression plasmid pCT44Ig-MGexpressing a concatameric fusion monomeric protein mgCTLA4-CTLA4/Fccontaining three glycosylation motif peptides according to the presentinvention;

[0044]FIG. 15 shows a result of SDS-PAGE of purified concatameric fusiondimeric proteins [TNFR1-TNFR1/Fc]₂ and [TNFR2-TNFR2/Fc]₂ under reducingor non-reducing conditions;

[0045]FIG. 16 is a graph showing inhibitory effect of the conventionalsimple fusion dimeric proteins [TNFR1/Fc]₂() and [TNFR2/Fc]₂(◯) and theconcatameric fusion dimeric proteins [TNFR1-RNFR1/Fc]₂(▾) and[TNFR2-TR2Fc]₂(∇) according to the present invention against cytotoxicactivity of TNF-alpha;

[0046]FIG. 17 is a graph showing inhibitory effect of the conventionalsimple fusion dimeric proteins [TNFR1/Fc]₂() and [TNFR2/Fc]₂(◯) and theconcatameric fusion dimeric proteins [TNFR1-RNFR1/Fc]₂(▾) and[TNFR2-TR2Fc]₂(∇) according to the present invention against cytotoxicactivity of TNF-beta;

[0047]FIG. 18 is a graph showing inhibitory effect of the conventionalsimple fusion dimeric protein [CD2/Fc]₂(), the known immunosuppressiveagent cyclosporin A (▾) and the concatameric fusion dimeric protein[CD2-CD2/Fc]₂(◯) according to the present invention on the proliferationof active T lymphocytes;

[0048]FIG. 19 is a graph showing inhibitory effect of the conventionalsimple fusion dimeric protein [CTLA4/Fc]₂(), the knownimmunosuppressive agent cyclosporin A (▾) and the concatameric fusiondimeric protein [CTLA4-CTLA4/Fc]₂ (◯) according to the present inventionon the proliferation of active T lymphocytes;

[0049]FIG. 20 is a graph showing blood half-life of the conventionalsimple fusion dimeric protein [TNFR1/Fc]₂(), the concatameric dimericprotein [TNFR1-TNFR1/Fc]₂ (◯) and a glycosylated concatameric fusiondimeric protein [mgTNFR1-TNFR1/Fc]₂ (∇) according to the presentinvention;

[0050]FIG. 21 is a graph showing blood half-life of the conventionalsimple fusion dimeric protein [CD2/Fc]₂(), the concatameric fusiondimeric protein [CD2-CD2/Fc]₂ (◯) and a glycosylated concatameric fusiondimeric protein [mgCD2-CD2/Fc]₂ (∇) according to the present invention;

[0051]FIG. 22 is a graph showing blood half-life of the conventionalsimple fusion dimeric protein [CTLA4/Fc]₂(), the concatameric fusiondimeric protein [CTLA4-CTLA4/Fc]₂ (◯) and a glycosylated concatamericfusion dimeric protein [mgCTLA4-CTLA4/Fc]₂ (∇) according to the presentinvention; and

[0052]FIG. 23 is a graph showing inhibitory effect of PBS () as acontrol, the conventional simple fusion dimeric proteins [TNFR1/Fc]₂ (▪)and [TNFR2/Fc]₂ (▴), and concatameric fusion dimeric proteins[TNFR1-TNFR1/Fc]₂ (x) and [TNFR2-TNFR2/Fc]₂ (Δ) according to the presentinvention on the induction of collagen-induced arthritis (CIA) in DBA/1mice.

BEST MODE FOR CARRYING OUT THE INVENTION

[0053] The present invention is generally directed to concatamericproteins, and more particularly, to immunoadhesion molecules.Immunoadhesion molecules are typically formed by fusion of the Fcfragment of immunoglobulin (Ig) to a ligand-binding region of a receptoror an adhesion molecule, and thus have a structure similar to that of anantibody. The typical immunoadhesion molecules known in the art have astructure of an antibody in which the variable region is substitutedwith a ligand-binding region of a receptor while retaining the Fcfragment. A wide variety of immunoadhesion molecules are suggested inthe literature. However, immunoadhesion molecules according to thepresent invention have different structure with the conventionalimmunoadhesion molecules, and there is also no prior art predicting ordescribing preparation of the immunoadhesion molecules according to thepresent invention.

[0054] Definition of Terms

[0055] For full understanding of the characteristic structure of theimmunoadhesion molecules according to the present invention, exactdefinitions of the terms used in the present invention are given asfollows. In general, all of the technical and scientific terms being notadditionally defined in the present invention have meanings commonlyused in the art. However, although having meanings commonly used in theart, the following terms are defined to give a clearer understanding oftheir meanings and make the scope of the present invention clear, asfollows.

[0056] The term “immunoglobulin”, as used herein, refers to proteinmolecules being produced in B cells and serving as antigen receptorsspecifically recognizing a wide variety of antigens. The molecules havea Y-shaped structure consisting of two identical light chains (L chains)and two identical heavy chains (H chains), in which the four chains areheld together by a number of disulfide bonds, including the disulfidebridge between the H chains at the hinge region. The L and H chainscomprise variable and constant regions. The L chain variable regionassociates with the H chain variable region, thus producing twoidentical antigen-binding regions. According to features of the constantregions of H chains, immunoglobulins (Ig) are classified into fiveisotypes, A (IgA), D (IgD), E (IgE), G (IgG) and M (IgM). Each subtypepossesses unique structural and biological properties. For example, IgGhas slightly different Fc structure, compared with other isotypes. Inaddition, IgG and IgA have a number of subtypes. For example, the humanIgG isotype has four subtypes, IgG1, IgG2, IgG3 and IgG4, which have γ1,γ2, γ3 and γ4 H chains, respectively. Biological functions ofimmunoglobulin molecules, such as complement activation, Fcreceptor-mediated phagocytosis and antigen-dependent cytotoxicity, aremediated by structural determinants (complementarity-determiningregions) in the Fc region of H chains. Such an Fc region of H chains isused for construction of dimeric proteins according to the presentinvention, and may be derived from all isotypes and subtypes ofimmunoglobulin as described above.

[0057] The term “Fc fragment of an immunoglobulin molecule”, as usedherein, refers to a fragment having no antigen-binding activity andbeing easily crystallized, which comprises a hinge region and CH2 andCH3 domains, and a portion responsible for binding of an antibody toeffector materials and cells. Therefore, the Fc fragment mentioned inthe present invention can be different from that described in someliteratures, but includes the hinge region. Such description of the Fcfragment is given to supply convenience in describing the presentinvention, and will be fully understood by those of ordinary skill inthe art with reference to the specification of the present invention andthe accompanying drawings.

[0058] The term “biologically active protein”, as used herein, refers toa protein, peptide or polypeptide having generally physiological orpharmaceutical activities, which retains a part of its native activitiesafter forming a concatamer or immunoadhesion molecule. The term“biological activity”, as used herein, is not limited in meaning tophysiological or pharmaceutical activities. For example, someconcatamers, such as those containing an enzyme can catalyze a reactionin an organic solvent. Similarly, some high-molecular weight fusionmolecules containing concanavalin A or an immunoglobulin molecule areuseful as diagnostic agents in laboratories.

[0059] Non-limiting examples of the protein, peptide or polypeptideinclude hemoglobin, serum proteins (e.g., blood factors including factorVII, VIII and factor IX), immunoglobulin, cytokines (e.g., interleukin),α-, β- and γ-interferon, colony-stimulating agent (e.g., G-CSF andGM-CSF), platelet-derived growth factor (PDGF), and phospholipaseactivating proteins (PLAPs). Other typical biological or therapeuticproteins include insulin, plant proteins (e.g., lectin and ricin), tumornecrosis factor (TNF) and its related alleles, growth factors (e.g.,tissue growth factors and endothelial growth factors such as TGFα orTGFβ), hormones (e.g., follicle-stimulating hormone, thyroid-stimulatinghormone, antidiuretic hormone, pigment-concentrating or dispersinghormones and parathyroid hormone, luteinizing hormone-releasing hormoneand its derivatives, calcitonin, calcitonin gene related peptide (CGRP),synthetic enkephalin, somatomedin, erythropoietin, hypothalamusreleasing factors, prolactin, chronic gonadotrophin, tissueplasminogen-activating agents, growth hormone-releasing peptide (GHRP),and thymic humoral factor (THF). The immunoglobulins include IgG, IgE,IgM, IgA, IgD and fragments thereof. Some proteins such as interleukin,interferon or colony-stimulating factor may be produced in anon-glycosylated form using DNA recombinant techniques. Thenon-glycosylated proteins may be useful as biologically active materialsin the present invention.

[0060] In addition, the biologically active materials useful in thepresent invention include any polypeptide, which has bioactivity invivo. Examples of the biologically active materials include peptides orpolypeptides, fragments of an antibody, single chain-binding proteins(see U.S. Pat. No. 4,946,778), binding molecules including fusionpolypeptides of antibodies or their fragments, polyclonal antibodies,monoclonal antibodies, and catalytic antibodies. Other examples of thebiologically active materials include allergen proteins, such asragweed, antigen E, honeybee venom, or allergen of mites.

[0061] In addition, the biologically active material useful in thepresent invention includes enzymes. Examples of the enzymes includecarbohydrate-specific enzymes, proteinases, oxidoreductases,transferases, hydrolases, lyases, isomerases, and ligases. In detail,non-limiting examples of the enzymes include asparaginase, arginase,arginine deaminase, adenosine deaminase, peroxide dismutase,endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosinedephosphatase, tyrosinase, and bilirubin oxidase. Examples of thecarbohydrate-specific enzymes include glucose oxidase, glucodase,galactosidase, glucocerebrosidase, and glucouronidase.

[0062] The term “proteins involving immune response”, as used herein,refers to all proteins mediating cell-to-cell signal transduction duringcellular or humoral immune response and thus activating or suppressingimmune response. Immunity is a process of protecting “self” from“non-self” such as bacteria or viruses. Immune response is largelydivided into cellular and humoral immune response, where T and Blymphocytes play the most important role. T cells, mainly mediatingcellular immune response, directly attack and kill virus-infected cellsor tumor cells, or help other immune cells by secreting cytokinesfunctioning to induce or activate immune response or inflammation. Bcells produce antibodies against non-self foreign materials (antigens)that enter a body, such as bacteria or viruses, and such immune responseis called cellular immune response. Cell-to-cell signal transduction isan essential process in both cellular and humoral immune responses, inwhich a signal molecule, that is, a ligand, interacts with a cellsurface receptor acting to transduce a specific signal into a cell.

[0063] Representative examples of the proteins involving the immuneresponse according to the present invention include cytokines, cytokinereceptors, adhesion molecules, tumor necrosis factor receptor (TNFR),enzymes, receptor tyrosine kinases, chemokine receptors, other cellsurface proteins, and soluble ligands. Non-limiting examples of thecytokines include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10,IL-12, IL17, TNF, TGF, IFN, GM-CSF, G-CSF, EPO, TPO, and M-CSF. Examplesof the cytokine receptors, but are not limited to, include growthhormone receptors (GHRs), IL-13R, IL-1R, IL-2R, IL-3R, IL-4R, IL-5R,IL-6R, IL-7R, IL-9R, IL-15R, TNFR, TGFR, IFNR (e.g., IFN-γ R α-chain andIFN-γ R β-chain), interferon-α R, -β R and -γ R, GM-CSFR, G-CSFR, EPOR,cMpl, gp130, and Fas (Apo 1). Non-limiting examples of the enzymesinclude influenza C hemaglutinin esterase and urokinase. The chemokinereceptors are exemplified by CCR1 and CXCR1-4. Examples of the receptortyrosine kinases, but are not limited to, include TrkA, TrkB, TrkC, Htk,REK7, Rse/Tyro-3, hepatocyte growth factor R, platelet-derived growthfactor R, and Flt-1. Examples of other cell surface proteins includesCD2, CD4, CD5, CD6, CD22, CD27, CD28, CD30, CD31, CD40, CD44, CD100,CD137, CD150, LAG-3, B7, B61, β-neurexin, CTLA-4, ICOS, ICAM-1,complement R-2 (CD21), IgER, lysosomal membrane gp-1, α2-microglobulinreceptor-related proteins, and sodium-releasing peptide R. Non-limitingexamples of the soluble ligands include IL-10, heregulin, andkeratinocyte growth factors.

[0064] Ligands for the proteins involving immune response according tothe present invention and use thereof are well known to those ofordinary skill in the art, as summarized in Tables 1 to 7, below. TABLE1 Proteins involving immune response: Adhesion molecules Adhesionmolecules Ligands Uses CD4 HIV gp120 Inhibition of in vivo HIVinfection; and identification of CD4 domain participating in ligandbinding L-Selectin GlyCAM-1, CD34 Prevention of neutrophile-mediatedlung damage; determination of position in tissues of a ligand byhistochemical staining; and isolation and cloning of ligands anddetermination of their properties E-Selectin Sialyl Lewis^(X) Preventionof neutrophile-mediated lung damage; and determination of thermodynamicproperties in ligand- binding P-Selectin Sialyl Lewis^(X) Prevention ofneutrophile-mediated lung damage; and study of functions of individualof amino acid residues in binding to cell surface ICAM-1 CD11a/CD18Phagocytosis of erythrocytes in malaria; inhibition of infection withrhinovirus; and anti-inflammation in diabetes ICAM-2 CD11a/CD18 Study ofactivation of T cells mediated by T cell receptor ICAM-3 CD11a/CD18Identification of receptor domains binding to a ligand VCAM-1 VLA-4Study of role of VLA-4 in T lympho- cyte migration to dermalinflammation sites LFA-3 CD2 Study of role of CD2 in costimulation of Tcells L1 Fibroblast growth Stimulation of nerve reproductionglycoprotein factor receptor after repair; and functional comparisonwith FGF

[0065] TABLE 2 Proteins involving immune response: Enzymes EnzymesLigands Uses Influenza C 9-0-acetylated Inactive enzyme used in study ofhemaglutinin sialic acid tissue-specific expression of ligands esteraseUrokinase Urokinase receptor Inactive enzyme developed to inhibit cancermetastasis by disturbing urokinase activation

[0066] TABLE 3 Proteins involving immune response: Cytokine receptorsCytokine receptors Ligands Uses IFN-γ R α-chain IFN-γ Inhibition ofIFN-mediated auto- immunity IFN-γ R β-chain IFN-γ Study of structure ofsubunits of a ligand-receptor complex IL1R IL-1 Inhibition ofIL-1-mediated inflammation IL4R IL-4 Identification of receptor domainsparticipating in ligand binding Erythropoietin R Erythropoietin Mapdesign of epitopes of anti- ligand antibodies cMp1 ThrombopoietinIsolation and cloning of ligands gp130 IL-6-IL6R Study of structure ofsubunits of a complex ligand-receptor complex

[0067] TABLE 4 Proteins involving immune response: Tumor necrosis factorreceptors TNF receptors Ligands Uses TNF R-1 TNF, Treatment of septicshock, rheumatoid lymphotoxin-α arthritis and other inflammatorydiseases; and identification of domains participating in ligand bindingTNF R-2 TNF, Inhibition of TNF-enriched HIV lymphotoxin-α replication;and prevention of collagen- induced arthritis in mice Lymphotoxin-Lymphotoxin-β Study of structure of subunits of cell β R surfacelymphotoxin-β Fas/Apo- Fas/Apo- Treatment of excessive apoptosis and1/CD95 1/CD95 ligand related diseases (e.g., AIDS); and resistance toapoptosis of lymphocytes and peripheral immune tolerance; roles of Fasligand in T cell-mediated cytotoxicity; and isolation and cloning ofligands CD27 CD27 ligand Isolation and cloning of ligands CD30 CD30ligand Isolation and cloning of ligands CD40 gp39 Isolation and cloningof ligands 4-1BB 4-1BB ligand Identification of tissues containingligands by histochemical staining; isolation and cloning of ligands; andStudy of structural determinant of potential ligand OX40 gp34 Isolationand cloning of ligands

[0068] TABLE 5 Proteins involving immune response: Receptor tyrosinekinases Receptor tyrosine kinases Ligands Uses TrkA, B, C NeutropinDetermination of properties of neutropin binding Htk Htk ligandIsolation and cloning of ligands REK7 AL-1 Isolation and cloning ofligands Rse/Tyro-3 Protein S, Gas6 Identification of ligands anddetermination of their properties Hepatocyte Hepatocyte growthIdentification of receptor growth factor R factor domains participatingin ligand binding Platelet-derived Platelet-derived Identification ofreceptor growth factor R growth factor domains participating in ligandbinding Flt-1 Vesicular endothelial Determination of properties ofgrowth factor (VEGF) ligand binding of receptors Flk-1/KDR VEGFEvaluation of selectivity of receptors for VEGF versus placenta growthfactor

[0069] TABLE 6 Proteins involving immune response: Other cell surfaceproteins Other cell surface proteins Ligands Uses B7 CD28 Study of Tcell stimulation by B cells B61 Eck Roles of Eck in inflammationβ-neurexin β-neurexin ligand Determination of properties of a signalsequence from β-neurexin CD2 LFA-3, CD48 Identification of ligands CD5CD5 ligand Study of T cell stimulation by B cells CD6 ALCAM Study ofbinding activities of cloned ligands CD22 CD45, other Identification ofligands; study on sialoglycoproteins roles of CD22 in T-B-cell inter-action; and determination of properties of binding determinants ofsialo-oligo sugar ligands CD28 B7, B7-2 Study of T cell stimulation by Bcells CD31 CD31 Identification of CD31 domains related to homotypebinding CD44 Hyaluronate Screening of tissues containing ligands byhistochemical staining; and determination of properties of structuraldeterminants of ligands Complement C3 fragment Inhibition of reactivityof antibody R-2 (CD21) to immunosuppressive and cancer therapeuticagents CTLA-4 B7 Identification of CTLA-4 as a secondary receptor of B7IgER IgE Inhibition of mast cell-binding of IgE as therapy of allergicdiseases Lisosome LAMP-1 ligand Design of epitope maps of anti- membranegp-1 ligand antibodies α2-microglobulin gp330 Determination of positionof receptor-bound ligands in tissues by histochemical proteins stainingSodium-releasing Sodium-releasing Design of epitope maps of anti-peptide R peptide ligand antibodies; and preparation of recombinantreceptors for structural study

[0070] TABLE 7 Proteins involving immune response: Soluble ligandsSoluble ligands Ligands Uses IL-2 IL-2R Extension of half-life of IL-2in the circulation system IL-10 IL-10R Therapy of septic shock andtransplan- tation rejection; and extension of half- life of IL-10 in thecirculation system Heregulin Her4/p180^(erbB4) Study of signaltransduction by Her4 Keratinocyte Keratinocyte Determination of positionof receptors growth factor growth factor R by histochemical staining

[0071] The term “soluble extracellular domain”, as used herein, refersto a portion exposed to the extracellular region of an integral membraneprotein penetrating the cell membrane comprising phospholipid, whereinthe integral membrane protein contains one or more transmembrane domainmade up predominantly of hydrophobic amino acids. Such an extracellulardomain mainly comprises hydrophilic amino acids, which are typicallypositioned at the surface of a folded structure of a protein, and thusis soluble in an aqueous environment. For most cell surface receptorproteins, extracellular domains serve to bind specific ligands, whileintracellular domains play an important role in signal transduction.

[0072] The term “concatamer-linked”, as used herein, refers to a statein which two soluble domains of biologically active proteins are linkedand thus form a long polypeptide.

[0073] The term “concatameric protein”, as used herein, means aconcatamer-linked protein. For example, the N-terminus of a solubleextracellular domain of a protein involving immune response is linked tothe C-terminus of an identical soluble extracellular domain of theprotein involving immune response, wherein the C-terminus of the formersoluble extracellular domain is linked to the hinge region of an Fcfragment of an immunoglobulin molecule. Thus, two identical solubleextracellular domains of a protein involving immune response form a longpolypeptide.

[0074] The term “simple fusion monomeric protein”, as used herein,refers to a fusion protein having a monomeric structure consisting of asingle polypeptide formed by linkage of a soluble extracellular domainof a protein involving immune response to the hinge region of an Fcfragment of an immunoglobulin molecule. A simple fusion monomericprotein may be designated “protein name/Fc” for convenience in thepresent invention. For example, a simple fusion monomeric proteinproduced by linkage of an soluble extracellular domain of TNFR1 proteininvolving immune response to an Fc fragment of an immunoglobulinmolecule is designated TNFR1/Fc. If desired, the origin of the Fcfragment may be also specified in the designation. For example, in thecase that the Fc fragment is derived from IgG1, the monomeric protein iscalled TNFR1/IgG1Fc.

[0075] The term “simple fusion dimeric protein”, as used herein, refersto a fusion protein having a dimeric structure, in which two simplefusion monomeric proteins are joined by formation of intermoleculardisulfide bonds at the hinge region. Such a simple fusion dimericprotein may be designated “[protein name/Fc]₂” for convenience in thepresent invention. For example, when fused by formation ofintermolecular disulfide bonds at the hinge region of two simple fusionmonomeric proteins produced by linkage of an soluble extracellulardomain of TNFR1 protein and an Fc fragment of an immunoglobulinmolecule, the resulting fusion protein having dimeric structure isdesignated [TNFR1/Fc]₂. In addition, the origin of the Fc fragment maybe specified in the designation, if desired. For example, in the casethat the Fc fragment is derived from IgG1, the dimeric protein isdesignated [TNFR1IgG1Fc]₂.

[0076] The term “concatameric fusion monomeric protein”, as used herein,refers to a fusion protein having a monomeric structure consisting of asingle polypeptide, in which the N-terminus of a soluble extracellulardomain of a protein involving immune response is linked to theC-terminus of an identical soluble extracellular domain of the proteininvolving immune response, wherein the C-terminus of the former solubleextracellular domain is linked to the hinge region of an Fc fragment ofan immunoglobulin molecule. A concatameric fusion monomeric protein maybe designated “protein name-protein name/Fc” for convenience in thepresent invention. For example, when an soluble extracellular domain ofTNFR1 of a simple fusion monomeric protein, produced by linkage of thesoluble extracellular domain of TNFR1 protein involving immune responseand an Fc fragment of an immunoglobulin molecule, is linked to anidentical soluble extracellular domain of TNFR1, the resultingconcatameric fusion monomeric protein is designated TNFR1-TNFR1/Fc. Ifdesired, the origin of the Fc fragment may be specified in thedesignation. For example, in the case that the Fc fragment is derivedfrom IgG1, the monomeric protein is designated TNFR1-TNFR1/IgG1Fc.

[0077] The term “concatameric fusion dimeric protein”, as used herein,refers to a fusion protein having a dimeric structure, in which twoconcatameric fusion monomeric proteins are fused by formation ofintermolecular disulfide bonds at the hinge region. A concatamericfusion dimeric protein may be designated “[protein name-proteinname/Fc]₂” for convenience in the present invention. For example, whentwo concatameric fusion monomeric proteins, each of which is produced bylinkage of a TNFR1 soluble extracellular domain of a simple fusionmonomeric protein to an identical soluble extracellular domain of TNFR1protein involving immune response, are fused by formation ofintermolecular disulfide bonds at the hinge region, the resulting fusionprotein having dimeric structure is designated [TNFR1-TNFR1/Fc]₂,wherein the simple fusion monomeric protein is formed by linkage of theTNFR1 soluble extracellular domain to an Fc fragment from animmunoglobulin molecule. If desired, the origin of the Fc fragment maybe specified in the designation. For example, in the case that the Fcfragment is derived from IgG1, the fusion protein is designated[TNFR1-TNFR1/IgG1Fc]₂.

[0078] The term “vector”, as used herein, means a DNA molecule servingas a vehicle capable of stably carrying exogeneous genes into hostcells. For useful application, a vector should be able to replicate,have a system for introducing itself into a host cell, and possessselectable markers. The exogeneous genes, for example, include, a DNAconstruct encoding a concatameric fusion monomeric protein.

[0079] The term “recombinant expression plasmid”, as used herein, refersto a circular DNA molecule carrying exogeneous genes operably linkedthereto to be expressed in a host cell. When introduced into a hostcell, the recombinant expression plasmid has the ability to replicateregardless of host chromosomal DNA, copy itself at a high copy number,and to produce heterogeneous DNA. As generally known in the art, inorder to increase the expression level of a transfected gene in a hostcell, the gene should be operably linked to transcription andtranslation regulatory sequences functional in a host cell selected asan expression system. Preferably, the expression regulation sequencesand the exogeneous genes may be carried in a single expression vectorcontaining. bacteria-selectable markers and a replication origin. Incase that eukaryotic cells are used as an expression system, theexpression vector should further comprise expression markers useful inthe eukaryotic host cells.

[0080] The term “operably linked”, as used herein, means an arrangementof elements of a vector, in which each element is capable of performingits innate function. Therefore, a control sequence operably linked to acoding sequence can influence expression of the coding sequence. Acontrol sequence acting to induce expression of a coding sequence doesnot have to be adjacent to the coding sequence. For example, when anintervening sequence is present between a promoter sequence and a codingsequence, the promoter sequence may still be “operably linked” to thecoding sequence.

[0081] Host cells used in the present invention may be prokaryotic oreukaryotic. In addition, host cells having high introduction efficiencyof foreign DNA and having high expression levels of an introduced genemay be typically used. Examples of the host cells useful in the presentinvention include prokaryotic and eukaryotic cells such as E. coli,Pseudomonas sp., Bacillus sp., Streptomyces sp., fungi or yeast, insectcells such as Spodoptera frugiperda (Sf9), animal cells such as Chinesehamster ovary cells (CHO) or mouse cells, African green monkey cellssuch as COS 1, COS 7, human embryonic kidney cells, BSC 1, BSC 40 or BMT10, and tissue-cultured human cells. When cloning a DNA constructencoding the fusion protein according to the present invention, hostcells are preferably animal cells. When using COS cells, since SV40large T antigen is expressed in COS cells, a plasmid carrying a SV 40replication origin may be present as a multicopy episome and thus allowshigh expression of an exogeneous gene. A DNA sequence introduced into ahost cell may be homogeneous or heterogeneous to the host cell, or ahybrid DNA sequence containing a homogenous or heterogeneous DNAsequence.

[0082] In order to express a DNA sequence encoding the concatamericfusion protein according to the present invention, a wide variety ofcombinations of host cells as an expression system and vectors may beused. Expression vectors useful for transforming eukaryotic host cellscontain expression regulation sequences from, for example, SV40, bovinepapillomavirus, adenovirus, adeno-associated viruses, cytomegalovirusand retroviruses. Expression vectors useful in bacterial host cellsinclude bacterial plasmids from E. coli, which are exemplified bypBluescript, pGEX2T, pUC, pCR1, pBR322, pMB9 and derivatives thereof,plasmids having a broad range of host cells, such as RP4, phage DNAs,exemplified by a wide variety of λ phage derivatives including λ gt10, λgt11 and NM989, and other DNA phages, exemplified by filamentoussingle-stranded DNA phages such as M13. Expression vectors useful inyeast cells include 2μ plasmid and derivatives thereof. Expressionvectors useful in insect cells include pVL 941.

[0083] The term “transformation”, as used herein, means introducing DNAinto a suitable host cell so that the DNA is replicable, either as anextrachromosomal element, or by chromosomal integration.

[0084] The term “transfection”, as used herein, refers to the taking upof an expression vector by a suitable host cell, whether any codingsequences are in fact expressed or not.

[0085] The term “signal sequence”, as used herein, means an amino acidsequence mediating transport of an expressed protein to the outside ofthe cell membrane, and is also called a “leader sequence”. Cell surfaceproteins or secretory proteins, which are transported to the outside ofthe cell membrane, have an N-terminal sequence typically cut by signalpeptidase in the cell membrane. Such a N-terminal sequence is called asignal sequence or signal peptide, or a leader sequence or leaderpeptide. Secretory (or transported) proteins or all proteins presentoutside of the cell membrane or in the extracellular environment have aspecific signal sequence. There is no specific homology between suchsignal sequences and same proteins have different signal sequencesaccording to their origin. Secondary structure or distribution ofnonpolar and charged residues is more important for proper function ofthe signal sequences than primary structures thereof. Although nothaving specific homology, the signal sequences share several commonfeatures, as follows. The signal sequences contain an N domain at theirN-termini, which is a hydrophilic region comprising one or morepositively charged residues, and an H-domain follows the N domain, whichis a somewhat long hydrophobic region. In the case of E. coli, thesignal sequence comprises about 18-30 amino acids. The N domain containsmany cationic amino acids such as Lys or Arg, and thus has a netpositive charge. Many hydrophobic amino acids such as Ala or Leu arefound in the H domain, and polar or charged amino acids such as Pro,Lys, Arg, Asn or Glu are rarely in the H domain. A large number of aminoacids such as Ala and Leu residues form an α-helical structure tofacilitate membrane penetration. A C domain is positioned between the Hdomain and an actually secreted portion of a protein. The C domain isless hydrophobic, and contains a sequence capable of being recognized bysignal peptidase such as LebB or LspA. There have been no reports aboutan exact site cleaved by the signal peptidase, but the signal peptidaseis typically known to mostly cleave behind the Ala-X-Ala sequence in theC domain. Preproteins containing the above-mentioned signal sequencearrive at the cell membrane through interaction with several proteins,and fold to their mature forms through cleavage of a specific region ofa signal peptide. Such a signal sequence is very important in strategiesto express a desired protein on the cell surface or in the extracellularenvironment. Foreign proteins and fusion proteins should be stablytransported to the extracellular environment at high efficiency.Typically, cell surface proteins having excellent secretory ability areuseful for cell surface expression of foreign proteins or fusionproteins, which typically have secretory signal sequences capable ofoffering excellent secretion efficiency.

[0086] Preparation of the Concatameric Fusion Dimeric Protein Accordingto the Present Invention

[0087] The concatameric fusion dimeric protein according to the presentinvention is generally prepared by (a) preparing a DNA constructencoding a simple fusion monomeric protein using a gene encoding an Fcfragment of an immunoglobulin molecule and a gene encoding a solubleextracellular domain of a protein involving immune response; (b)inserting by polymerase chain reaction (PCR) a recognition sequence of arestriction enzyme into the prepared simple fusion monomericprotein-encoding DNA construct and an identical gene to the geneencoding a soluble extracellular domain of a protein involving immuneresponse, respectively; (c) cleaving the recognition sequence of arestriction enzyme in the simple fusion monomeric protein-coding DNAconstruct and the gene encoding a soluble extracellular domain of aprotein involving immune response using the restriction enzymerecognizing the recognition sequence; (d) ligating the cleaved DNAfragments using ligase to produce a DNA construct encoding aconcatameric fusion monomeric protein (see, FIG. 2); (e) operablylinking the prepared DNA construct encoding a concatameric fusionmonomeric protein to a vector to produce a recombinant expressionplasmid; (f) transforming or transfecting a host cell with therecombinant expression plasmid; and (g) culturing the transformant ortransfectant under conditions suitable for expression of the DNAconstruct encoding a concatameric fusion monomeric protein and thenisolating and purifying a concatameric fusion dimeric protein ofinterest.

[0088] A DNA fragment encoding a soluble extracellular domain of aprotein involving immune response is produced by PCR using a primercontaining a recognition sequence of a specific restriction enzyme and asequence encoding a leader sequence, and a primer containing anantisense sequence encoding the 3′ end of the soluble extracellulardomain and a portion of the 5′ end of a specific region of Fc fragmentof an immunoglobulin molecule.

[0089] A DNA fragment encoding a specific region of the Fc fragment ofan immunoglobulin molecule is produced by PCR using a primer having asequence encoding a portion of the 3′ end of the soluble extracellulardomain of the protein involving immune response and a sequence encodingthe 5′ end of the specific region of the Fc fragment of animmunoglobulin molecule, and another primer having an antisense sequenceencoding a recognition sequence of a specific restriction enzyme and the3′ end of a specific region of the Fc fragment of an immunoglobulinmolecule.

[0090] The DNA fragment encoding a soluble extracellular domain of aprotein involving the immune response and the DNA fragment encoding aspecific region of Fc fragment of an immunoglobulin molecule, asdescribed above, are mixed in a test tube. After denaturation, the DNAis re-annealed. Then, a complete double-stranded DNA fragment isproduced by polymerization using DNA polymerase at the 3′ end of eachDNA hybrid. Using the resulting double-stranded DNA fragment, anotherpolymerase chain reaction (PCR) is carried out with the primer having asequence encoding a soluble extracellular domain of a protein involvingimmune response and the primer encoding the 3′ end of a specific regionof the Fc fragment of an immunoglobulin molecule, in order to amplify aimmunoglobulin fusion gene comprising a sequence corresponding to theDNA fragment encoding a soluble extracellular domain of a proteininvolving immune response and a sequence corresponding to the DNAfragment encoding a specific region of the Fc fragment of animmunoglobulin molecule.

[0091] An recognition sequence of a restriction enzyme is introduced byPCR into the amplified immunoglobulin fusion gene and the DNA fragmenthaving a sequence encoding a soluble extracellular domain of a proteininvolving the immune response. The recognition sequence is then cleavedwith the restriction enzyme and the cleaved regions are ligated usingligase, thus producing a concatameric immunoglobulin fusion gene.

[0092] The immunoglobulin fusion gene may further include a signalsequence to stimulate extracellular secretion of a protein encodedthereby. For example, the CTLA-4 molecule contains a unique leadersequence having highly hydrophilic redundancy at its N-terminus, andwhich is abnormally long and highly water-soluble (Harper, K. et al., J.Immunol. 147:1037-1044; and Brunet, J. F. Nature 328:267-270, 1987).Generally, most cell surface proteins or secretory proteins have aleader sequence comprising 20-24 highly hydrophobic amino acids at theirN-termini. However, the CTLA-4 molecule used in the present inventioncomprises a total of 37 residues: 16 hydrophilic amino acids at itsN-terminus, and 21 highly hydrophobic amino acids typical in itstransmembrane regions. In the conventional method of preparing CTLA4Igfusion proteins, the leader sequence of the CTLA-4 molecule wassubstituted with a leader sequence of oncostatin M (Linsley, P. S. etal., J. Exp. Med. 174:561-569, 1991) or IL-6 (Yamada, A, et al.,Microbiol. Immunol. 40:513-518, 1996). The present inventorsdemonstrated that a CTLA-4 molecule containing a leader sequence havinga “MRTWPCTLLFFIPVFCKA” sequence instead of the amino acid sequenceconsisting of 16 amino acids, “ACLGFQRHKAQKNLAA”, is preferable, and thesecretion of an expressed protein to the extracellular environment iseasily achieved, as disclosed in International Pat. Publication No.WO98/31820.

[0093] A recombinant expression plasmid is prepared by inserting theimmunoglobulin fusion gene into a vector, and then introduced to a hostcell to produce a transformant or transfectant. A concatameric fusiondimeric protein of interest may be obtained by culturing thetransformant or transfectant cell and isolating and purifying aconcatameric fusion protein.

[0094] A host cell useful for preparation of the concatameric fusiondimeric protein according to the present invention is preferablyselected from among bone marrow cell lines, CHO cells, monkey COS cells,human embryonic kidney 293 cells, and baculovirus-infected insect cells.A polypeptide of interest, produced in such an expression system, issecreted to culture medium as an inclusion body. Then, the concatamericfusion dimeric protein can be purified by affinity chromatography usinga protein A or protein G column. In fact, effective mammalian expressionsystems and such purification systems are very useful in expressingproteins involving immune response in a dimeric form, and isolation ofsuch proteins.

[0095] Preparation of the Glycosylated Concatameric Fusion DimericProtein According to the Present Invention

[0096] Secretory proteins produced in eukaryotic cells as host cells aremodified by glycosylation. Glycosylation is known to influence in vivostability and functionality as well as physical properties of a protein.Therefore, a preferred aspect of the present invention includesfacilitating production of a concatameric fusion dimeric protein ofinterest using recombinant DNA techniques and the above-mentioned animalcell lines as host cells, and linking additional sugar chains to asoluble extracellular domain of a protein involving immune response.

[0097] Two glycosylation patterns are known. One is O-linkedglycosylation, in which an oligosaccharide is linked to a serine orthreonine residue, and the other is N-linked glycosylation, in which anoligosaccharide is linked to asparagine residue. N-linked glycosylationoccurs at a specific amino acid sequence, particularly, Asn-X-Ser/Thr,wherein X is any amino acid excluding proline. N-linked oligosaccharidehas a structure distinct from O-linked oligosaccharide, and glycosylatedresidues found in the N-linked type also differ from the O-linked type.For example, N-acetylgalactosamine is invariably linked to serine orthreonine in O-linked oligosaccharide, while N-acetylglucosamine islinked to asparagines in all of N-linked oligosaccharides. The O-linkedoligosaccharides generally contain only 1-4 sugar residues. In contrast,the N-linked oligosaccharides comprise 5 or more sugar residues,essentially including N-acetylglucosamine and mannose.

[0098] In accordance with the present invention, to allow additionalO-linked or N-linked glycosylation, one or more nucleotides in a DNAsequence encoding a soluble extracellular domain of a protein involvingimmune response are altered, and the resulting DNA is expressed in asuitable animal host cell to induce glycosylation using the host system.In accordance with an aspect of the present invention, the glycosylatedconcatameric fusion dimeric protein according to the present inventionmay be prepared by altering a DNA sequence encoding a solubleextracellular domain of a protein involving immune response to induce orincrease N-linked glycosylation by adding the sequence Asn-X-Ser/Thr.

[0099] Alteration of a DNA sequence to introduce glycosylation may beperformed according to the conventional method common in the art. In apreferred aspect of the present invention, to protect the concatamericfusion protein, especially the two soluble extracellular domains, fromattack of intercellular proteinases and thus increase its half-life inserum, a DNA construct encoding a multiglycosylated concatameric fusionmonomeric protein may be prepared using PCR, which introducesmultiglycosylation sites to the joint region between two solubleextracellular domains. In a specific aspect of the present invention,glycosylation motif peptide sequences may be introduced into theconcatameric fusion protein, as follows. A DNA fragment is prepared byperforming PCR using a primer encoding a leader sequence of a solubleextracellular domain and EcoRI restriction site, and an antisense primerin which a portion of a nucleotide sequence encoding a portion of the 3′end of a first soluble extracellular domain and a portion of the 5′ endof a second soluble extracellular domain is substituted withglycosylation motif sequences. Another DNA fragment is prepared byperforming PCR using a primer in which a portion of a nucleotidesequence encoding a portion of the 3′ end of a first solubleextracellular domain and a portion of the 5′ end of a second solubleextracellulular domain is substituted with glycosylation motifsequences, and an antisense primer encoding the 3′ end of Fc portion ofIgG1 and XbaI restriction site. Then, secondary PCR is carried out in atest tube using the two DNA fragments.

[0100] In accordance with an embodiment of the present invention, thesoluble extracellular domains useful in the present invention includesoluble extracellular domains of TNFR1, TNFR2, CD2 and CTLA-4. Theirapplication will be described in detail with reference to accompanyingfigures, sequence listing and examples.

[0101] Tumor necrosis factor-alpha (TNF-α), which is known as thehormone cachectin, and tumor necrosis factor-beta (TNF-β), which is alsoknown as lymphotoxin, are multifunctional cytokines, inducinginflammation, cellular immune response, septicemia, cytotoxicity,cachexia, rheumatoid arthritis, inflammation-related diseases(Tartaglia, L. A. et al., Immunol. Today 13:151,1992), and antiviralreaction (Butler, P., Peptide Growth Factor II, 1990, Springer-Verlag,Berlin, pp.39-70). Such actions of TNF-α and TNF-β, including cytotoxicactivity, originate from their binding to TNF receptors in a trimericform (Eck, M. J. et al., J. Biol. Chem. 267:2119, 1992). As TNFreceptors, 55 kDa-type I (TNFR1 or p55) and about 75 kDa-type II (TNFR2or p75) are known (Smith, C. A. et al., Science 248:1019, 1990;Loetscher, H. et al., Cell 61:351, 1990; and Schall et al., Cell 61:361,1990). The two receptors have similar affinity for TNF-α and TNF-β(Schall et al., Cell 61:361, 1990). Immunoglobulin fusion proteins ofsuch soluble receptors have effects of inhibiting the action of TNF-αand TNF-β by inhibiting binding of TNF-α and TNF-β to their receptors onthe cell surface, which is known to be effective in reducingTNF-dependent inflammation.

[0102] Among cell surface antigens regulating immune response, thecostimulatory molecule CD2 and CTLA-4, inducing secondary stimulation togive sufficient activation of T cells, when being in a soluble form,also can be used for therapy of diverse immunological diseases accordingto the same method as TNF receptors. Immune response is accomplished bybinding of cell surface antigen molecules of antigen presenting cells(APC) to specific receptors of T lymphocytes, that is, T lymphocytes andleukocyte-function-antigen molecules of APC, and when a costimulatorysignal as a secondary signal is not produced during antigen-presenting,T lymphocytes are removed by apoptosis or inhibition of clonalactivation. CD2 is a leukocyte-function-antigen on T lymphocytes,binding to LFA-3 on APC, and participates in adhesion and costimulationof leukocytes, as well as stimulating T cell activation throughcostimulation with CD28. CTLA-4 is expressed after activation of Tlymphocytes, and its expression level is increased in the resting phase.CTLA-4 has a binding affinity to the B7 molecule of APC over 20 timeshigher than that of CD28, and transduces signals inhibiting T lymphocyteactivation after binding to B7.

[0103] In a specific aspect of the present invention, there are provideda concatameric fusion monomeric protein TNFR1-TNFR1/Fc, designated bySEQ ID NO: 6; a concatameric fusion monomeric protein TNFR2-TNFR2/Fc,designated by SEQ ID NO: 8; a concatameric fusion monomeric proteinCD2-CD2/Fc, designated by SEQ ID NO: 18; and a concatameric fusionmonomeric protein CTLA4-CTLA4/Fc, designated by SEQ ID NO: 20.

[0104] In another specific aspect of the present invention, there areprovided a DNA construct (TNFR1-TNFR1-IgG) encoding a concatamericfusion monomeric protein TNFR1-TNFR1/Fc, designated by SEQ ID NO: 5; aDNA construct (TNFR2-TNFR2-IgG) encoding a concatameric fusion monomericprotein TNFR2-TNFR2/Fc, designated by SEQ ID NO: 7; a DNA construct(CD2-CD2-IgG) encoding a concatameric fusion monomeric proteinCD2-CD2/Fc, designated by SEQ ID NO: 17; and a DNA construct(CTLA4-CTLA4-IgG) encoding a concatameric fusion monomeric proteinCTLA4-CTLA4/Fc, designated by SEQ ID NO: 19.

[0105] In a further specific aspect of the present invention, there areprovided a recombinant expression plasmid pTR11Ig-Top10′ operably linkedto a DNA construct encoding a concatameric fusion monomeric proteinTNFR1-TNFR1/Fc, designated by SEQ ID NO: 5; a recombinant expressionplasmid pTR22Ig-Top10′ operably linked to a DNA construct encoding aconcatameric fusion monomeric protein TNFR2-TNFR2/Fc, designated by SEQID NO: 7; a recombinant expression plasmid pCD22Ig operably linked to aDNA construct encoding a concatameric fusion monomeric proteinCD2-CD2/Fc, designated by SEQ ID NO: 17; and a recombinant expressionplasmid pCT44Ig operably linked to a DNA construct encoding aconcatameric fusion monomeric protein CTLA4-CTLA4/Fc, designated by SEQID NO: 19. The recombination expression plasmids are deposited in KoreanCulture Center of Microorganisms (KCCM) and are assigned accession Nos.KCCM-10288, KCCM-10291, KCCM-10402 and KCCM-10400, respectively. TheKCCM deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure.

[0106] In a further specific aspect of the present invention, there areprovided a mammalian host cell (e.g., TR11Ig-CHO) transformed ortransfected with a recombinant expression plasmid pTR11Ig-Top10′operably linked to a DNA construct encoding a concatameric fusionmonomeric protein TNFR1-TNFR1/Fc, designated by SEQ ID NO: 5; amammalian host cell (e.g., TR22Ig-CHO) transformed or transfected with arecombinant expression plasmid pTR22Ig-Top10′ operably linked to a DNAconstruct encoding a concatameric fusion monomeric proteinTNFR2-TNFR2/Fc, designated by SEQ ID NO: 7; a mammalian host celltransformed or transfected with a recombinant expression plasmid pCD22Igoperably linked to a DNA construct encoding a concatameric fusionmonomeric protein CD2-CD2/Fc, designated by SEQ ID NO: 17; and amammalian host cell transformed or transfected with a recombinantexpression plasmid pCT44Ig operably linked to a DNA construct encoding aconcatameric fusion monomeric protein CTLA4-CTLA4/Fc, designated by SEQID NO: 19. Chinese hamster ovary cell line TR11Ig-CHO transfected withthe recombinant expression plasmid pTR11Ig-Top10′ and Chinese hamsterovary cell line TR22Ig-CHO transfected with the recombinant expressionplasmid pTR22Ig-Top10′ are deposited in KCCM and are assigned accessionNos. KCLRF-BP-00046 and KCLRF-BP-00049, respectively. The KCCM depositwill be maintained under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure.

[0107] In a still further specific aspect of the present invention,there are provided a concatameric fusion monomeric proteinmgTNFR1-TNFR1/Fc containing glycosylation motif peptides, designated bySEQ ID NO: 10; a concatameric fusion monomeric protein mgTNFR2-TNFR2/Fccontaining glycosylation motif peptides, designated by SEQ ID NO: 12; aconcatameric fusion monomeric protein mgCD2-CD2/Fc containingglycosylation motif peptides, designated by SEQ ID NO: 22; and aconcatameric fusion monomeric protein mgCTLA4-CTLA4/Fc containingglycosylation motif peptides, designated by SEQ ID NO: 24.

[0108] In a still further specific aspect of the present invention,there are provided a DNA construct encoding a concatameric fusionmonomeric protein mgTNFR1-TNFR1/Fc containing glycosylation motifpeptides, designated by SEQ ID NO: 9; a DNA construct encoding aconcatameric fusion monomeric protein mgTNFR2-TNFR2/Fc containingglycosylation motif peptides, designated by SEQ ID NO: 11; a DNAconstruct encoding a concatameric fusion monomeric protein mgCD2-CD2/Fccontaining glycosylation motif peptides, designated by SEQ ID NO: 21;and a DNA construct encoding a concatameric fusion monomeric proteinmgCTLA4-CTLA4/Fc containing glycosylation motif peptides, designated bySEQ ID NO: 23. In order to produce a glycosylation motif peptide, aprimer set (forward and reverse primers) is designed, which arecomplementary to a nucleotide sequence corresponding to the joint regionbetween soluble extracellular domains of concatameric fusion proteins ofTNFR/Fc, CD2/Fc and CTLA4/Fc, as well as containing codons encodingasparagine (N) (ATT and AAC) or codons encoding serine (S) and threonine(T) (TCC; and ACC, ACG and ACA, respectively), with which any codon inthe concatameric fusion protein gene may be substituted. When designingthe primer, selection of one among a plurality of amino acid sequencesmay be determined depending on a condition allowing minimum substitutionof the nucleotide sequence and melting temperature (T_(m)) of eachprimer.

[0109] In a still further specific aspect of the present invention,there are provided a recombinant expression plasmid pTR11Ig-MG operablylinked to a DNA construct encoding a concatameric fusion monomericprotein mgTNFR1-TNFR1/Fc containing glycosylation motif peptides,designated by SEQ ID NO: 9; a recombinant expression plasmid pTR22Ig-MGoperably linked to a DNA construct encoding a concatameric fusionmonomeric protein mgTNFR2-TNFR2/Fc containing glycosylation motifpeptides, designated by SEQ ID NO: 11; a recombinant expression plasmidpCD22Ig-MG operably linked to a DNA construct encoding a concatamericfusion monomeric protein mgCD2-CD2/Fc containing glycosylation motifpeptides, designated by SEQ ID NO: 21,; and a recombinant expressionplasmid Pct44Ig-MG operably linked to a DNA construct encoding aconcatameric fusion monomeric protein mgCTLA4-CTLA4/Fc containingglycosylation motif peptides, designated by SEQ ID NO: 23. Therecombination expression plasmids are deposited in Korean Culture Centerof Microorganisms (KCCM) and are assigned accession Nos. KCCM-10404,KCCM-10407, KCCM-10401 and KCCM-10399, respectively. The KCCM depositwill be maintained under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure.

[0110] In a still further specific aspect of the present invention,there are provided a mammalian host cell transformed or transfected witha recombinant expression plasmid pTR11Ig-MG operably linked to a DNAconstruct encoding a concatameric fusion monomeric proteinmgTNFR1-TNFR1/Fc containing glycosylation motif peptides, designated bySEQ ID NO: 9; a mammalian host cell transformed or transfected with arecombinant expression plasmid pTR22Ig-MG operably linked to a DNAconstruct encoding a concatameric fusion monomeric proteinmgTNFR2-TNFR2/Fc containing glycosylation motif peptides, designated bySEQ ID NO: 11; a mammalian host cell transformed or transfected with arecombinant expression plasmid pCD22Ig-MG operably linked to a DNAconstruct encoding a concatameric fusion monomeric protein mgCD2-CD2/Fccontaining glycosylation motif peptides, designated by SEQ ID NO: 21;and a mammalian host cell transformed or transfected with a recombinantexpression plasmid Pct44Ig-MG operably linked to a DNA constructencoding a concatameric fusion monomeric protein mgCTLA4-CTLA4/Fccontaining glycosylation motif peptides, designated by SEQ ID NO: 23.

[0111] The concatameric fusion dimeric proteins of the present inventionmay be isolated from culture medium after culturing the transformants ortransfectants according to the present invention. The concatamericfusion dimeric proteins may participate in immune response, as describedin Table 1, above, and are thus useful as therapeutic agents, diagnosticagents and laboratory tools according to the kinds of the protein, andtheir use is well known to those of ordinary skill in the art. Inparticular, when being used as therapeutic agents, the concatamericfusion dimeric proteins may be applied at an therapeutically effectiveamount common in the art, and it will be understood that such an amountmay vary depending on diverse factors including activity of the usedcompound, patient's age, body weight, health state, sex and diet,administration time, administration route, combination of drugs, andpathogenic state of a specific disease to be prevented or treated. Inaddition, when being used as therapeutic agents, it will be understoodthat the concatameric fusion dimeric proteins according to the presentinvention may be applied by the typical methods and routes foradministration of proteins involving immune response, which are known tothose of ordinary skill in the art.

[0112] The present invention will be explained in more detail withreference to the following examples in conjunction with the accompanyingdrawings. However, the following examples are provided only toillustrate the present invention, and the present invention is notlimited to them. For convenience in describing the present invention,information on DNA constructs, recombinant expression plasmids andtransformed cell lines, which are prepared according to the Examples,below, and the used primers and accession numbers is summarized inTables 8 and 9, below. TABLE 8 Information on DNA constructs andaccession Nos. SEQ ID No. Deposition of genes Deposition of cell linesDNA construct name DNA Protein Designation Accession No. DesignationAccession No. TNFR1-IgG 1 2 INFR2-IgG 3 4 TNFR1-TNFR1-IgG 5 6pTR11Ig-Top10′ KCCM 10288 TR11Ig- KCLRF-BP- CHO 00046 TNFR2-TNFR2-IgG 78 pTR22Ig-Top10′ KCCM 10291 TR22Ig- KCLRF-BP- CHO 00049mgTNFR1-TNFR1-IgG 9 10 pTR11Ig-MG KCCM 10404 mgTNFR2-TNFR2-IgG 11 12PTR22Ig-MG KCCM 10407 CD2-IgG 13 14 CTLA4-IgG 15 16 CD2-CD2-IgG 17 18pCD22Ig KCCM 10402 CTLA4-CTLA4-IgG 19 20 pCT44Ig KCCM 10400mgCD2-CD2-IgG 21 22 pCD22Ig-MG KCCM 10401 mgCTLA4-CTLA4-IgG 23 24pCT44Ig-MG KCCM 10399

[0113] TABLE 9 Information for primers SEQ Primer name ID No.Description Oligo TNFR-EDF- 25 Containing 5′ end of the extracellularEcoRI domain of TNFR1 and an EcoRI site Oligo TNFR-EDR- 26 Reverseprimer containing 3′ end of the IgGh extracellular domain of TNFR1 andthe hinge region of IgG Oligo IgG1-T1F 27 Containing 5′ end of the hingeregion of IgG and 3′ end of TNFR1 Oligo IgG1-R-XbaI 28 Reverse primercontaining 3′ end of the hinge region of IgG and a XbaI site OligoTNFR2-EDF- 29 Containing 5′ end of the extracellular EcoRI domain ofTNFR2 and an EcoRI site Oligo TNFR2-EDR- 30 Reverse primer containing 3′end of the IgGh extracellular domain of TNFR2 and the hinge region ofIgG Oligo IgG1-T2F 31 Containing 5′ end of the hinge region of IgG and3′ end of TNFR2 Oligo TNFR1-CF- 32 Containing 5′ end of theextracellular BamHI domain of TNFR1 and a BamHI site; and used forpreparation of a concatamer Oligo TNFR1-NR- 33 Reverse primer containing3′ end of the BamHI extracellular domain of TNFR1 and a BamHI site; andused for preparation of a concatamer Oligo TNFR2-CF- 34 Containing 5′end of the extracellular BamHI domain of TNFR2 and a BamHI site; andused for preparation of a concatamer Oligo TNFR2-NR- 35 Reverse primercontaining 3′ end of the BamHI extracellular domain of TNFR2 and a BamHIsite; and used for preparation of a concatamer Oligo mgTNFR1- 36 Primerfor mutagenesis, containing a TNFR1-IgG-F sequence capable of insertingglyco- sylation sites into the joint region of TNFR1-TNFR1, andsequences corresponding to 3′ end and 5′ end of TNFR1; and used forpreparation of a MG (multiglycosylation) form Oligo mgTNFR1- 37 Reverseprimer for mutagenesis, TNFR1-IgG-R containing a sequence capable ofinserting glycosylation sites into the joint region of TNFR1-TNFR1, andsequences corresponding to 3′ end and 5′ end of TNFR1; and used forpreparation of a MG form Oligo mgTNFR2- 38 Primer for mutagenesis,containing a TNFR2-IgG-F sequence capable of inserting glyco- sylationsites into the joint region of TNFR2-TNFR2, and sequences correspondingto 3′ end and 5′ end of TNFR2; and used for preparation of a MG formOligo mgTNFR2- 39 Reverse primer for mutation, containing TNFR2-IgG-R asequence capable of inserting glyco- sylation sites into the jointregion of TNFR2-TNFR2, and sequences corresponding to 3′ end and 5′ endof TNFR2; and used for preparation of a MG form Oligo CD2F-EcoRI 40Containing 5′ end of the extracellular domain of CD2 and a EcoRI siteOligo CD2R-RstI 41 Containing 3′ end of the extracellular domain of CD2and a PstI site Oligo IgG-F-PstI 42 Containing 5′ end of the hingeregion of IgG and a PstI site Oligo CTLA4F-EcoRI 43 Containing 5′ end ofthe extracellular domain of CTLA-4 and a EcoRI site Oligo CTLA4R-PstI 44Containing 3′ end of the extracellular domain of CTLA-4 and a PstI siteOligo CD2-NT-F 45 Containing 5′ end of the extracellular domain of CD2;and used for preparation of a concatamer Oligo CD2-CT-R 46 Reverseprimer containing 3′ end of the extracellular domain of CD2; and usedfor preparation of a concatamer Oligo CTLA4-NT-F 47 Containing 5′ end ofthe extracellular domain of CTLA-4; and used for preparation of aconcatamer Oligo CTLA4-CT-R 48 Reverse primer containing 3′ end of theextracellular domain of CTLA-4; and used for preparation of a concatamerOligo mgCD2-CD2- 49 Used for preparation of a MG (multi- IgG-Fglycosylation) form of CD2-CD2-IgG Oligo mgCD2-CD2- 50 Reverse primerused for preparation of a IgG-R MG (multiglycosylation) form ofCD2-CD2-IgG Oligo mgCTLA4- 51 Used for preparation of a MG (multi-CTLA4-IgG-F glycosylation) form of CTLA4-CTLA4- IgG Oligo mgCTLA4- 52Reverse primer used for preparation of CTLA4-IgG-R a MG(multiglycosylation) form of CTLA4-CTLA4-IgG

EXAMPLE 1 Human TNFR

[0114] A. Manufacture of a DNA Construct Encoding Simple FusionMonomeric Protein of TNFR1/Fc (FIG. 1 and FIG. 5)

[0115] a. DNA Fragment Encoding Soluble Extracellular Domain of TNFR1

[0116] A fusion gene encoding soluble extracellular domain of type Ihuman TNF receptor (TNFR1, p55) and Fc fragment of human immunoglobulinG1 was constructed by the Polymerase Chain Reaction (PCR) methoddescribed in the prior art (Holten et al., Biotechniques 8:528, 1990).

[0117] A DNA fragment encoding soluble extracellular domain of TNFR1 wasconstructed by PCR using a primer (the sequence of nucleotide of SEQ IDNO: 25) with EcoRI restriction site and the sequence encoding leadersequence (the sequence of amino acids 1-20 of SEQ ID NO: 2), and anantisense primer (the sequence of nucleotide of SEQ ID NO: 26) with thesequence encoding a part of 3′ ends of the said soluble extracellulardomain of TNFR1 (TNFR1-ED) and 5′ ends of the hinge region ofimmunoglobulin G1 (IgG1). The template cDNA for this reaction wasconstructed by reverse transcription PCR (RT-PCR) of mRNA extracted frommonocyte (T lymphocyte) of healthy adults.

[0118] After blood of healthy adults was extracted and diluted to 1:1with RPMI-1640 (Gibco BRL, USA), the layer of T lymphocyte which formedat upper part was obtained by density gradient centrifugation usingFicoll-hypaque (Amersham, USA). In order to make the concentration ofthe cell to 5×10⁵ cells/ml, the cell was washed with RPMI-1640 for 3times, and RPMI-1640 culture media containing 10% Fetal Bovine Serum(FBS, Gibco BRL, USA) was added, then cultured at 37° C. for two days inthe 5% CO₂ incubator after adding leukoagglutinin to 3.5 ug/ml(Pharmacia, USA).

[0119] The mRNAs were purified using Tri-Reagent (MRC, USA) mRNApurification kit. First, 2×10⁷ of human T lymphocyte was washed withPhosphate Buffered Saline (PBS, pH7.2) for 3 times, and then 1 ml ofTri-Reagent was mixed for several times to dissolve RNA. After adding0.2 ml of chloroform to this tube and mixing thoroughly, this tube wasincubated at room temperature (RT) for 15 min, then centrifuged at15,000 rpm, 4° C. for 15 min. The upper part of the solution wastransferred to a 1.5 ml tube, and 0.5 ml of isopropanol was added, andthen centrifuged at 15,000 rpm, 4° C. for 15 min. After the supernatantwas discarded, the pellet was resuspended with 1 ml of 3° distilledwater treated with 75% ethanol-25% DEPC (Sigma, USA), and thencentrifuged at 15,000 rpm, 4° C. for 15 min. After the supernatant wasremoved completely and dried in the air to remove ethanol residue, RNAwas resuspended with 50 μl of 3° distilled water treated with DEPC.

[0120] The primary cDNA was synthesized by mixing 2 μg of purified mRNAand 1 μl of oligo dT (dT30, Promega, USA) primer to 10 μM in 1.5 mltube, heating at 70° C. for 2 min, and cooling in ice for 2 min. Afterthat, this mixture was added with 200 U of M-MLV reverse transcriptase(Promega, USA), 10 μl of 5×reaction buffer (250 mM Tris-HCl, pH 8.3, 375mM KCl, 15 mM MgCl₂, and 50 mM DTT), 1 μl of dNTP (10 mM each, Takara,Japan), and DEPC-treated 3° distilled water to 50 μl, then reacted at42° C. for 1 hour.

[0121] b. DNA Fragment Encoding Fc Fragment of Immunoglobulin

[0122] A DNA fragment encoding Fc fragment of immunoglobulin G1 wasconstructed by PCR using a primer (the sequence of nucleotide of SEQ IDNO: 27) with the sequence encoding a part of 3′ ends of the said solubleextracellular domain of TNFR and 5′ end of the hinge region ofimmunoglobulin G1 (IgG1), and an antisense primer (the sequence ofnucleotide of SEQ ID NO: 28) with XbaI restriction site and the sequenceencoding 3′ ends of IgG1 Fc. The template cDNA for this reaction wasconstructed by RT-PCR of mRNA extracted from peripheral blood cell (Blymphocyte) of convalescent patients with pyrexia of unknown origin.

[0123] c. DNA Construct Encoding Simple Fusion Monomeric Protein ofTNFR1/Fc

[0124] After DNA fragment encoding soluble extracellular domain of TNFR1and DNA fragment encoding Fc fragment of immunoglobulin produced asdescribed above were mixed in the same tube, complementary bindingbetween the common sequence (the sequence including 3′ end of solubleextracellular domain of TNFR1 and 5′ end of IgG1 hinge region) wasinduced. Using this mixture as a template, DNA construct including DNAfragment encoding soluble extracellular domain of TNFR1 and DNA fragmentencoding IgG1 Fc fragment was amplified by PCR using a primer (thesequence of nucleotide of SEQ ID NO: 25) with the sequence encoding 5′end of TNFR1 and another primer (the sequence of nucleotide of SEQ IDNO: 28) with the sequence encoding 3′ end of IgG1 Fc. The constructedgene included a leader sequence to faciliate secretion of protein afterexpression.

[0125] d. Cloning of the DNA Construct Encoding Simple Fusion MonomericProtein of TNFR1/Fc

[0126] DNA construct encoding simple fusion monomeric protein ofTNFR1/Fc as described above was restricted with EcoRI and XbaI, andcloned by inserting into a commercially available cloning vector,pBluescript KS II (+) (Stratagene, USA), at EcoRI/XbaI site. Thesequence of a total coding region was identified by DNA sequencing (SEQID NO: 1). This produced fusion protein was designated TNFR1/Fc assimple fusion monomeric protein, and the elliptical shape shown in FIG.1 represents the structure of a primary expression product of the fusiongene. The deduced amino acid sequence of simple fusion monomeric ofTNFR1/Fc corresponded to SEQ ID NO: 2.

[0127] B. Manufacture of a DNA Construct Encoding Simple FusionMonomeric Protein of TNFR2/Fc (FIG. 1 and FIG. 5)

[0128] a. DNA Fragment Encoding Soluble Extracellular Domain of TNFR2

[0129] A fusion gene encoding soluble extracellular domain of type IIhuman TNF receptor (TNFR2, p75) and Fc fragment of human immunoglobulinG1 was constructed by the same method as that of TNFR1/Fc.

[0130] A DNA fragment encoding soluble extracellular domain of TNFR2 wasconstructed by PCR using a primer (the sequence of nucleotide of SEQ IDNO: 29) with EcoRI restriction site and the sequence encoding leadersequence (the sequence of amino acids 1-22 of SEQ ID NO: 4), and anantisense primer (the sequence of nucleotide of SEQ ID NO: 30) with thesequence encoding a part of 3′ ends of said soluble extracellular domainof TNFR2 (TNFR2-ED) and 5′ ends of the hinge region of immunoglobulin G1(IgG1). The template cDNA for this reaction was constructed by RT-PCR ofmRNA extracted from monocyte (T lymphocyte) of healthy adults.

[0131] b. DNA Construct Encoding Simple Fusion Monomeric Protein ofTNFR2/Fc

[0132] After DNA fragment encoding soluble extracellular domain of TNFR2and DNA fragment encoding Fc fragment of immunoglobulin G1 produced asdescribed above were mixed in the same tube, complementary bindingbetween the common sequence (the sequence including 3′ end of solubleextracellular domain of TNFR2 and 5′ end of IgG1 hinge region) wasinduced. Using this mixture as a template, DNA construct including DNAfragment encoding soluble extracellular domain of TNFR2 and encoding andDNA fragment encoding IgG1 Fc fragment was amplified by PCR using aprimer (the sequence of nucleotide of SEQ ID NO: 29) with the sequenceencoding 5′ end of TNFR2 and another primer (the sequence of nucleotideof SEQ ID NO: 28) with the sequence encoding 3′ end of IgG1 Fc. Theconstructed gene includes a sequence to faciliate secretion of proteinafter expression.

[0133] c. Cloning of the DNA Construct Encoding Simple Fusion MonomericProtein of TNFR2/Fc

[0134] DNA construct encoding simple fusion monomeric protein ofTNFR2/Fc as described above was restricted with EcoRI and XbaI, andcloned by inserting into a commercially available cloning vector,pBluescript KS II (+) (Stratagene, USA), at EcoRI/XbaI site. Thesequence of a total coding region was identified by DNA sequencing (SEQID NO: 3). This produced fusion protein was designated TNFR2/Fc assimple fusion monomeric protein, and the elliptical shape shown in FIG.1 represents the structure of a primary expression product of the fusiongene. The deduced amino acid sequence of simple fusion monomeric ofTNFR2/Fc corresponded to SEQ ID NO: 4.

[0135] C. Manufacture of a DNA Construct Encoding Concatameric FusionMonomeric Protein of TNFR1-TNFR1/Fc (FIG. 2 and FIG. 5)

[0136] In order to manufacture a fusion gene comprising the concatamericshape in soluble extracellular domain of TNFR1, i.e. the DNA constructencoding concatameric fusion monomeric protein of TNFR1-TNFR1/Fc, BamHIrestriction site was inserted respectively into the sequence of solubleextracellular domain of TNFR1 and DNA construct as produced as aboveencoding simple fusion monomeric protein of TNFR1/Fc by PCR, and thenregions of each fragments restricted by BamHI were linked by ligase. TheDNA construct, encoding simple fusion monomeric protein of TNFR1/Fcproduced as above, was used as the template of this reaction.

[0137] The fragment of the soluble extracellular domain of TNFR1 withBamHI restriction site at 3′ end was amplified by PCR using a primercorresponding to the nucleotide of SEQ ID NO: 25 and another primercorresponding to the nucleotide sequence of SEQ ID NO: 33, and the otherfragment of simple fusion monomeric protein of TNFR1/Fc with BamHIrestriction site at 5′ end was amplified by PCR using a primercorresponding to the nucleotide of SEQ ID NO: 28 and another primercorresponding to the nucleotide sequence of SEQ ID NO: 32, respectively.PCR was performed by adding 1 μl of primary cDNA, 2 U of Pfu DNApolymerase (Stratagene, USA), 10 μl of 10×reaction buffer [200 mMTris-HCl, pH 8.75, 100 mM (NH₄)₂SO₄, 100 mM KCl, 20 mM MgCl₂], 1%Triton™ X-100, 1 mg/ml BSA, 3 μl primer 1 (10 μM), 3 μl primer 2 (10μM), 2 μl dNTP (10 mM each), and 3° distilled water to 100 μl. Thereaction condition was as follows; 94° C., 5 min; 95° C., 1 min; 58° C.,1 min 30 see; 72° C., 1 min for 31 cycles; and 72° C., 15 min to makePCR product with complete blunt end.

[0138] After electrophorized on 0.8% agarose gel, the PCR product waspurified by Qiaex II gel extraction kit (Qiagen, USA). The purified PCRproduct was restricted by BamHI and extracted by phenol-chloroformextraction methods. Subsequently, two kinds of DNA fragments restrictedby BamHI were linked by ligase.

[0139] D. Manufacture of a DNA Construct Encoding Concatameric FusionMonomeric Protein of TNFR2-TNFR2/Fc (FIG. 2 and FIG. 5)

[0140] After a BamHI restriction site was inserted respectively into thesequence of the soluble extracellular domain of TNFR21 and the DNAconstruct produced as described above encoding simple fusion monomericprotein of TNFR2/Fc by PCR, a DNA construct encoding concatameric fusionmonomeric protein of TNFR2-TNFR2/Fc was manufactured by linking theregions of each fragments restricted by BamHI by ligase.

[0141] A fragment of soluble extracellular domain of TNFR2 with BamHIrestriction site at 3′ end was amplified using a primer correspondingthe sequence of SEQ ID NO: 34 and SEQ ID NO: 35. PCR was performed asthat of TNFR1, except that a DNA construct encoding simple fusionmonomeric protein of SEQ ID NO: 3 produced as above was used as atemplate. The PCR product was purified by the method as that of TNFR1.

[0142] E. DNA Construct Encoding Concatameric Fusion Monomeric Proteinof TNFR1-TNFR1/Fc with Glycosylation Motif.

[0143] A DNA fragment was manufactured by PCR using an antisense primer(the sequence of nucleotide of SEQ ID NO: 37) with the sequence encodingthe part (the sequence of nucleotide 565-591 of SEQ ID NO: 5) of 3′ endof the first soluble extracellular domain of TNFR1, except the sequenceof hydrophobic peptide region (the sequence of amino acid 197-216 of SEQID NO: 6) at the junction of soluble extracellular domain of TNFR1 andthe part (the sequence of nucleotide 649-681 of SEQ ID NO: 5) of 5′ endof the second soluble extracellular domain of TNFR1, and another primer(the sequence of nucleotide of SEQ ID NO: 25) with the sequence encodingEcoRI restriction site and leader sequence.

[0144] In addition, the total four amino acid sequences encodingglycosylation site (the sequence of amino acids 189-191, 192-194,198-200, and 204-206 of SEQ ID NO: 10) were inserted by manufacturingthe primer as above (the sequence of nucleotide of SEQ ID NO: 36 and 37)corresponding the substitution of the nucleotide 565-567 (CTG, Leu),574-576 (ACG, Thr), 652-654 (CTA, Leu), and 670-672 (AGA, Arg) of SEQ IDNO: 5 with the nucleotide of AAC (Asn, N); the nucleotide of 571-573(TGC, Cys) and 580-582 (TTG, Leu) of SEQ ID NO: 5 with the nucleotide ofACC (Thr, T); the nucleotide of 658-660 (GAC, Asp) with the nucleotideof TCC (Ser, S).

[0145] In this reaction, the gene (the nucleotide of SEQ ID NO: 5)encoding concatameric shape of TNFR1-TNFR1/Fc was used as a template.During the primary PCR, only the half of the antisense primer wasinduced to bind the gene encoding concatameric shape of TNFR1-TNFR1/Fcused as a template, and, as chain reaction was proceeding, the unboundpart to the template was induced to form a complete double-stranded DNAby polymerase, and then this was capable of producing the DNA fragmentwith state of linkage of the sequence of 5′ end encoding the part of thesecond soluble extracellular domain and the sequence of 3′ end encodingTNFR1 extracellular domain including leader sequence. Therefore, a partof the sequence of 5′ end encoding the second soluble extracellulardomain has the function that was capable of binding to the second DNAfragment as follows.

[0146] The second DNA fragment was manufactured by PCR using a primer(the sequence of nucleotide of SEQ ID NO: 36) with the sequence encodingthe part (the sequence of nucleotide 565-591 of SEQ ID NO: 5) of 3′ endof the first soluble extracellular domain of TNFR1 and the part (thesequence of nucleotide 649-681 of SEQ ID NO: 5) of 5′ end of the secondsoluble extracellular domain of TNFR1, and an antisense primer (thesequence of nucleotide of SEQ ID NO: 28) with the sequence encoding aXbaI restriction site and 3′ end of IgG1 Fc. This reaction was alsoperformed as described above, that is, only the half of antisense primerwas induced to bind the template, and consequently, DNA fragment likethat described above had the sequence encoding 5′ end of TNFR1extracellular including the part of 3′ end of the first solubleextracellular domain.

[0147] Subsequently, resulting from two kinds of DNA fragments as PCRdescribed as above were mixed in the same tube, induced to bind betweencommon sequences, and fused by PCR using primers (the sequence ofnucleotide of SEQ ID NO: 25 and 28) encoding 5′ and 3′ end of eachconcatameric genes, and the product was designated mgTNFR1-TNFR1-IgG.

[0148] F. DNA Construct Encoding Concatameric Fusion Monomeric Proteinof TNFR2-TNFR2/Fc with Glycosylation Motif.

[0149] A DNA fragment was manufactured by PCR using an antisense primer(the sequence of nucleotide of SEQ ID NO: 39) with the sequence encodingthe part (the sequence of nucleotide 586-606 of SEQ ID NO: 7) of 3′ endof first soluble extracellular domain of TNFR2, except the sequence ofhydrophobic peptide region (the sequence of amino acid 203-263 of SEQ IDNO: 8) at the junction of soluble extracellular domain of TNFR2 and thepart (the sequence of nucleotide 790-807 of SEQ ID NO: 7) of 5′ end ofsecond soluble extracellular domain of TNFR2, and another primer (thesequence of nucleotide of SEQ ID NO: 29) with the sequence encodingEcoRI restriction site and leader sequence.

[0150] In addition ,the total two amino acid sequences encodingglycosylation site (the sequence of amino acids 199-201 and 206-208 ofSEQ ID NO: 12) were inserted by manufacturing the primer as describedabove (the sequence of nucleotide of SEQ ID NO: 38 and 39) correspondingto the substitution of the nucleotide 595-597 (GTC, Val) and 799-801(GGG, Gly) SEQ ID NO: 7 with the nucleotide of AAC (Asn, N).

[0151] In this reaction, the gene (the nucleotide of SEQ ID NO: 7)encoding concatameric shape of TNFR2-TNFR2/Fc was used as a template.During the primary PCR, only the half of antisense primer was induced tobind the gene encoding concatameric shape of TNFR2-TNFR2/Fc used as atemplate, and, as the chain reaction was proceeding, the unbound part tothe template was induced to form a complete double-stranded DNA bypolymerase, and thus this was capable of producing the DNA fragment witha state of linkage of the sequence of 5′ end encoding the part of thesecond soluble extracellular domain and the sequence of 3′ end encodingTNFR2 extracellular domain including the leader sequence. Therefore, apart of the sequence of 5′ end encoding the second soluble extracellulardomain has the function that was capable of binding to the second DNAfragment as follows.

[0152] The second DNA fragment was manufactured by PCR using a primer(the sequence of nucleotide of SEQ ID NO: 38) with the sequence encodingthe part (the sequence of nucleotide 586-606 of SEQ ID NO: 7) of 3′ endof the first soluble extracellular domain of TNFR2 and the part (thesequence of nucleotide 790-807 of SEQ ID NO: 7) of 5′ end of the secondsoluble extracellular domain of TNFR2, and an antisense primer (thesequence of nucleotide of SEQ ID NO: 28) with the sequence encoding aXbaI restriction site and 3′ end of IgG1 Fc. This reaction was alsoperformed, that is, only the half of antisense primer was induced tobind the template, and consequently, DNA fragment like that describedabove had the sequence encoding 5′ end of TNFR2 extracellular includingthe part of 3′ end of first soluble extracellular domain.

[0153] Subsequently, resulting from two kinds of DNA fragments as PCRproduced as above were mixed in the same tube, induced to bind betweencommon sequences, and fused by PCR using primers (the sequence ofnucleotide of SEQ ID NO: 29 and 28) encoding 5′ and 3′ end of eachconcatameric genes, and the product was designated mgTNFR2-TNFR2-IgG.

[0154] G. Cloning of DNA Constructs Encoding Concatameric FusionMonomeric Protein of TNFR-TNFR/Fc and Their Glycosylated Forms

[0155] DNA constructs encoding concatameric fusion monomeric protein ofTNFR-TNFR/Fc and their glycosylated forms as above were cloned byinserting into pBluescript KS II (+) (Stratagene, USA) at EcoRI/XbaIsite. These produced fusion proteins were designated TNFR1-TNFR1/Fc andTNFR2-TNFR2/Fc as concatameric fusion monomeric protein, and designatedmgTNFR1-TNFR1/Fc and mgTNFR2-TNFR2/Fc as their glycosylated forms. Thededuced amino acid sequences corresponded to SEQ ID NO: 6, 8, 10,and 12.

[0156] After 10 μg of pBluescript KS II (+) (Stratagene, USA) used as avector was mixed with 15 U of EcoRI, 15 U of XbaI, 5 μl of 10× reactionbuffer (100 mM Tris-HCl, pH 7.5, 100 mM MgCl₂, 10 mM DTT, 500 nM NaCl),5 μl of 0.1% BSA (Takara, Japan), and 3° distilled water to 50 μl, DNAwas restricted by incubation at 37° C. for 2 hrs. After electrophorizedon 0.8% agarose gel, the PCR product was purified by Qiaex II gelextraction kit (Qiagen, USA).

[0157] After 100 ng of pBluescript KS II (+) (Stratagene, USA)restricted by EcoRI and XbaI was mixed with 20 ng of PCR productrestricted by the restriction enzyme, 0.5 U of T4 DNA ligase (Amersham,USA), 1 μl of 10× reaction buffer (300 mM Tris-HCl, pH 7.8, 100 mMMgCl₂, 100 mM DTT, 10 mM ATP) and 3° distilled water were added to 10μl, and the mixture was incubated in the water bath at 16° C. for 16hrs. E. coli Top10 (Novex, USA) was made to competent cell by the methodof rubidium chloride (RbCl, Sigma, USA) and transformed, then spread onthe solid LB media including 50 μg/ml of ampicillin (Sigma, USA) andincubated at 37° C. for 16 hrs. Formed colonies were inoculated in 4 mlof liquid LB media including 50 μg/ml of ampicillin and incubated at 37°C. for 16 hrs. Plasmid was purified by the method of alkaline lysisaccording to Sambrook et al. (Molecular cloning, Cold Spring HarborLaboratory press, p1.25-1.31, p1.63-1.69, p7.26-7.29, 1989) from 1.5 mlof that, and the existence of cloning was confirmed by the restrictionof EcoRI and XbaI.

[0158] The sequence of a total coding region was identified by the DNAsequencing method of dideoxy chain termination method (Sanger et al.,Proc. Natl. Acad. Sci., 74:5483, 1977) as follows. The DNA sequencingreaction was performed according to the manual using a plasmid purifiedby alkaline lysis method as described above and Sequenase™ ver 2.0(Amersham, USA). After the reaction mixture as above was loaded on 6%polyacrylamide gel and electrophorized for 2 hrs at constant voltage of1,800˜2,000 V and 50° C., DNA sequence was identified by exposing toX-ray film (Kodak, USA) after the gel was dried out.

EXAMPLE 2 AND 3 CD2 and CTLA4

[0159] DNA fragments encoding soluble extracellular domain of CD2 andCTLA4 were constructed by PCR using a primer [CD2(the sequence ofnucleotide of SEQ ID NO: 40), and CTLA4(the sequence of nucleotide ofSEQ ID NO: 43)] with EcoRI restriction site and the coding sequence [CD2(the sequence of nucleotide of SEQ ID NO: 13), and CTLA4 (the sequenceof nucleotide of SEQ ID NO: 15)] encoding the leader sequence [CD2(thesequence of amino acid 1-24 of SEQ ID NO: 14), and CTLA4(the sequence ofamino acid 1-21 of SEQ ID NO: 16)], and an antisense primer [CD2(thesequence of nucleotide of SEQ ID NO: 41), and CTLA4(the sequence ofnucleotide of SEQ ID NO: 44)] with PstI restriction site and thesequence [CD2(the sequence of nucleotide of SEQ ID NO: 13), andCTLA4(the sequence of nucleotide of SEQ ID NO: 15)] encoding 3′ end ofthe soluble extracellular domain of the proteins as described above. Thetemplate cDNA for this reaction was constructed by reverse transcriptionPCR (RT-PCR) of mRNA extracted from the monocyte (T lymphocyte) ofhealthy adults.

[0160] Also, a DNA fragment encoding Fc fragment of immunoglobulin G1was constructed by PCR using a primer (the sequence of nucleotide of SEQID NO: 42) with PstI restriction site and the sequence encoding 5′ endsof constant region of IgG1, and an antisense primer (the sequence ofnucleotide of SEQ ID NO: 28) with XbaI restriction site and the sequenceencoding 3′ ends of IgG1 Fc. The template cDNA for this reaction wasconstructed by RT-PCR of mRNA extracted from peripheral blood cell (Blymphocyte) of convalescent patients with unknown fever.

[0161] Subsequently, both DNA fragment encoding soluble extracellulardomain of CD2 and CTLA4 and DNA fragment encoding Fc fragment ofimmunoglobulin G1 produced as described above were restricted by PstI,and then the simple dimeric shape of CD2/Fc and CTLA4/Fc genes wereconstructed by linkages using T4 DNA ligase. The constructed genesincluded a leader sequence to faciliate secretion of protein afterexpression.

[0162] DNA constructs as described above were restricted by restrictionenzyme of EcoRI and XbaI, and cloned by inserting into a commerciallyavailable cloning vector, pBluescript KS II (+) (Stratagene, USA) atEcoRI/XbaI site. The sequence of a total coding region was identified byDNA sequencing (SEQ ID NO: 13 and 15). These produced fusion proteinswere designated CD2/Fc and CTLA4/Fc, and the deduced amino acidsequences of these corresponded to SEQ ID NO: 14 and 16.

[0163] PCR was performed by adding 1 μl of primary cDNA, 2 U of Pfu DNApolymerase (Stratagene, USA), 10 μl of 10× reaction buffer [200 mMTris-HCl, pH 8.75, 100 mM (NH₄)₂SO₄, 100 mM KCl, 20 mM MgCl₂], 1%Triton™ X-100, 1 mg/ml BSA, 3 μl primer 1 (10 μM), 3 μl primer 2 (10μM), 2 μl dNTP (10 mM each), and 3° distilled water to 100 μl. Thereaction condition was as follows; 94° C., 5 min; 95° C., 1 min; 58° C.,1 min 30 sec; 72° C., 1 min for 31 cycles; and 72° C., 15 min to makePCR product with complete blunt end.

[0164] The fusion genes with concatameric shape of CD2-CD2/Fc andCTLA4-CTLA4/Fc were constructed as follows.

[0165] In order to manufacture fusion gene comprising the concatamericshape in soluble extracellular domain of CD2 and CTLA4, the sequences ofsoluble extracellular domain of CD2 and CTLA4 were inserted by blunt-endligation using ligase at the junction between extracellular domain andimmunoglobulin of fusion genes in the shape of simple dimer with bluntend, using PstI restriction enzyme and T4 DNA polymerase. Specifically,DNA constructs were constructed by PCR using a primer [CD2(the sequenceof nucleotide of SEQ ID NO: 13) and CTLA4(the sequence of nucleotide ofSEQ ID NO: 48)] with the coding sequence [CD2(the sequence of nucleotideof SEQ ID NO: 13) and CTLA4(the sequence of nucleotide of SEQ ID NO:15)] encoding the end of leader sequence [CD2(the sequence of amino acid25 of SEQ ID NO: 14) and CTLA4(the sequence of amino acid 22 of SEQ IDNO: 16)] of soluble extracellular domain, and an antisense primer[CD2(SEQ ID NO: 46) and CTLA4(SEQ ID NO: 48)] with the sequence [CD2(thesequence of nucleotide of SEQ ID NO: 13) and CTLA4(the sequence ofnucleotide of SEQ ID NO: 15)] encoding 3′ end of soluble extracellulardomain as above. The simple fusion monomeric genes [CD2/Fc (the sequenceof nucleotide of SEQ ID NO: 13) and CTLA4/Fc (the sequence of nucleotideof SEQ ID NO: 15)] described as above were used as the template of thisreaction.

[0166] Also, CD2/Fc and CTLA4/Fc, which were inserted in pBluescript KSII (+) in the shape of simple monomeric form, were made to have 3′overhang end using the restriction enzyme of PstI. The cut end of 3′overhang was partially deleted to form a blunt end by treating T4 DNApolymerase. In order to manufacture fusion genes in the shape ofconcatamer in soluble extracellular domain, the soluble extracellulardomains of CD2 and CTLA4 produced by PCR as described above were clonedby inserting into cut ends of simple monomeric gene made as blunt end.These produced fusion proteins were designated CD2-CD2/Fc andCTLA4-CTLA4/Fc as concatameric fusion monomeric protein, and theirdeduced amino acid sequences corresponded SEQ ID NO: 18 and 20,respectively.

[0167] The concatameric fusion genes in the shape of multiglycosylatedform were constructed as follows.

[0168] The glycosylation mofit was inserted by secondary PCR with mixingin the same tube of a DNA fragment produced by PCR using a primerincluding EcoRI restriction site and the soluble extracellular domainwith leader sequence, and an antisense primer with the sequence encodingthe part of 3′ end of the first soluble extracellular domain ofconcatameric shape of fusion gene and the part of 5′ end of the secondsoluble extracellular domain with the nucleotide of substitutedglycosylation motif; and other DNA fragment produced by PCR using aprimer with the sequence encoding the part of 3′ end of the firstsoluble extracellular domain of concatameric shape of fusion gene andthe part of 5′ end of the second soluble extracellular domain with thenucleotide of substituted glycosylation motif, and an antisense primerwith the sequence encoding 3′ end of Fc fragment of immunoglobulin G1and XbaI restriction site.

[0169] In the case of concatameric fusion gene of CD2/Fc and CTLA4/Fc,the glycosylation motif was inserted by PCR using modified primers asthe same methods as that of TNFR/Fc described as above, but it wasdifferent from the case of TNFR/Fc that the amino acid sequence ofbinding to soluble extracellular domain of CD2 and CTLA4 was retained asthe same.

[0170] In the process of multiglycosylatin of the concatameric fusionprotein of CD2/Fc and CTLA4/Fc, the case of CD2/Fc was completed byinserting the total two glycosylation motif peptide region (the sequenceof amino acid of 200-202 and 206-208 of SEQ ID NO: 22) using amanufactured primer including the substitution of the nucleotide of598-600 (CCT, Pro) and 616-618 (GAG, Glu) of SEQ ID NO: 17 with AAT(Asn, N), and the case of CTLA4/Fc was completed by inserting the totalthree glycosylation motif peptide region (the sequence of amino acid of136-138, 142-144, and 147-149 of SEQ ID NO: 24) using a manufacturedprimer(SEQ ID NO: 51 and 52) including the substitution of thenucleotide of 403-405 (GTA, Val) and 424-426 (CCA, Pro) of SEQ ID NO: 19with AAT (Asn, N); the nucleotide of 409-411 (GAT, Asp) and 445-447(GTG, Val) with ACA (Thr, T) and ACG (Thr, T), respectively. Theseproduced fusion proteins were designated mgCD2-CD2/Fc andmgCTLA4-CTLA4/Fc as concatameric fusion monomeric protein, and theirdeduced amino acid sequences corresponded to SEQ ID NO: 22 and 24,respectively.

EXAMPLE 4 Expression and Purification of Simple/Concatameric FusionDimeric Protein of TNFR/Fc

[0171] In order to express the fusion proteins in CHO-K1 cell (ATCCCCL-61, Ovary, Chinese hamster, Cricetulus griseus), after pBluescriptKS II (+) plasmid DNA including TNFR/Fc fusion gene was purified fromtransformed E. coli, an animal cell expression vectors were constructedas TNFR/Fc fragment produced by restriction using EcoRI and XbaI wasinserted at EcoRI/XbaI site of an animal cell expression vector, pCR™3(Invitrogen, USA) plasmid. And these were designated plasmidpTR11-Top10′ and plasmid pTR22-Top10′, and deposited as accessionnumbers of KCCM 10288 and KCCM 10291, respectively, at Korean CultureCenter of Microorganisms (KCCM) on Jul. 10. 2001.

[0172] Transfection was performed by mixing either the plasmidpTR11-Top10′ or plasmid pTR22-Top10′ DNA including TNFR/Fc fusion genesas described above with the reagent of Lipofectamin™ (Gibco BRL, USA).CHO-K1 cells with the concentration of 1˜3×10⁵ cells/well wereinoculated in 6-well tissue culture plate (Nunc, USA), and incubated to50˜80% in 10% FBS-DMEM media, then the DNA-liposome complex, which wasreacted for 15˜45 min with 1˜2 μg of either the plasmid pTR11-Top10′ orplasmid pTR22-Top10′ DNA including TNFR/Fc fusion genes as describedabove and 2-25 μl of Lipofectamin™ (Gibco BRL, USA), were added to thecell culture plate in the serum-free DMEM media. After incubation for 5hrs, DMEM media with 20% serum was added and cells were incubatedfurther for 18˜24 hrs. After primary transfection, cells were incubatedfor 3 weeks in 10% FBS-DMEM media with 1.5 mg/ml of Geneticin (G418,Gibco BRL, USA), and formed colonies was selected for amplifiedincubation. The expression of fusion proteins was analyzed by ELISAusing a peroxidase labeled goat anti-human IgG (KPL, USA).

[0173] ELISA was performed as follows. First, 1 mg/ml of a peroxidaselabeled goat anti-human IgG (KPL, USA) was diluted to 1:2,000 with 0.1Msodium bicarbonate, 100 μl of that was aliquoted into 96-well flexibleplate (Falcon, USA) and sealed with plastic wrap, then incubated at 4°C. over 16 hrs to be coated on the surface of the plate. After this, itwas washed for 3 times with washing buffer (0.1% Tween-20 in 1×PBS) anddilution buffer (48.5 ml 1XPBS, 1.5 ml FBS, 50 ul Tween-20), and thenwas aliquoted to 1801. After 20 μl of culture supernatant was dropped inthe first well, then serially diluted using a micropipette, and 0.01μg/μl of human immunoglobulin G (Sigma, USA) as the positive control andthe culture media of untransfected CHO K-1 cell as the negative wasequally diluted. After dilution, 96-well ELISA plate (Falcon, USA) waswrapped with aluminum foil and incubated at 37° C. for 1 hr 30 min,washed for 3 times with washing buffer. Peroxidase conjugated goatanti-human IgG (KPL, USA) was diluted to 1:5,000 with dilution buffer,aliquoted to 100 μl, wrapped with aluminum foil, and reacted at 37° C.for 1 hr. After reaction, this plate was washed for 3 times, colorizedusing TMB microwell peroxidase substrate system (KPL, USA) and existenceof expression was confirmed by measurement of absorbance at 655 nmwavelength using microplate reader (Bio-Rad, Model 550, Japan).

[0174] Transfectants manufactured as above were designated TR11g-CHO andTR21Ig-CHO and deposited as accession numbers of KCLRF-BP-00046 andKCLRF-BP-00049, respectively, at Korean Cell Line Research Foundation(KCLRF) on Jul. 7. 2001. And adaptation for transfectants as describedabove to one of the serum free media, CHO-S-SFM II (Gibco BRL, USA), wasproceeded to purify the proteins produced by those transfectants asfollows. After about 3×10⁵ of cells were inoculated into the 6-wellplate, cells were cultured at 5% CO2, 37° C. for over 16 hrs to adhere,and it was checked under a microscope that cells were adhered at about30˜50% area of the plate, then cells were cultured in a media consistingof 10% FBS DMEM and CHO-S-SFM II in the ratio of 8:2. After culturing 3times serial passage at this ratio, it was cultured 3 times at the ratioof 6:4; 3 times at 4:6; 3 times at 3:7; 3 times at 2:8; 3 times at 1:9;and finally cultured in 100% CHO-S-SFM II media. And the level ofexpression was measured by ELISA.

[0175] After these transfectant cells were cultured on a large scale inCHO-S-SFM II, the supernatants including each fusion proteins werecentrifuged at 200×g for 12 min to remove cell debris, and proteins werepurified by the method using HiTrap protein A column (Amersham, USA) asfollows. After 20 mM of sodium phosphate (pH 7.0, Sigma, USA) was passedat the velocity of 1 ml/min for 2 min, 10 ml of supernatant was passedat the same velocity to bind fusion protein to protein A. After 20 mM ofsodium phosphate (pH 7.0) was passed at the same velocity for 2 min towash, 500 μl of the extracts were serially fractionated in a 1.5 ml tubeas 0.1M of citric acid (pH 3.0, Sigma, USA) was passed at the the samevelocity for 3 min. This was adjusted to pH 7.0 using 1M of Tris (pH11.0, USB, USA), the existence of fusion proteins in tube was confirmedthrough ELISA as described above. The purified proteins wereconcentrated by centrifugation at 2000×g, 4° C. for 30 min usingCentricon 30 (Amicon, USA)

EXAMPLE 5 SDS-PAGE of Purified TNFR1-TNFR1/Fc and TNFR2-TNFR2/Fc (FIG.15)

[0176] Proteins purified using protein A column were electrophorized bythe method of SDS-PAGE in reducing condition added by DTT, reducingreagent (which destroy disulfide bond), and in a non-reducing conditionexcluding DTT. The result of the estimation of molecular weight onSDS-PAGE is shown in Table 10. It was possible to confirm that TNFR/Fcproteins were the shape of a dimer in the cell. The molecular weightdeduced from the amino acid sequence of TNFR1-TNFR1-Ig was about 70 kDa,and was estimated as about 102 kDa on SDS-PAGE. As this difference couldbe regarded as a general phenomenon which generate on theelectrophoresis of glycoproteins, this feature seemed to occurr as theresult from decrease in mobility on the electrophoresis by the site ofglycosylation. TABLE 10 Molecular weight of TNFR-TNFR/Fc on theSDS-PAGE. Molecular weight (kDa) Proteins Reducing conditionNon-reducing condition TNFR1-TNFR1/Fc 102 200 TNFR2-TNFR2/Fc 115 220

EXAMPLE 6 Experiment of Neutralization Effect of Simple/ConcatamericFusion Dimeric TNFR/Fc Fusion Proteins on the Cytotoxicity of TNFα andTNFβ

[0177] An L929 cell [ATCC, Mus musculus (mouse), NCTC clone 929(derivative of strain L; L-929; L cell) was used for testing the effectof TNFR/Fc fusion protein on the inhibition of cytotoxicity induced byTNFα and TNFβ. This analysis was based on the TNFR activity ofinhibiting cytotoxicity induced by TNF (Scallon et al., Cytokine 7:759,1995).

[0178] L929 cells were inoculated to be 3×10⁴ cells/well in 96-wellplates, and incubated at 37° C. for 24 hrs in a CO₂ incubator.Subsequently, actinomycin D (Sigma, USA) was added to 3 μg/ml, and cellswere incubated for 16˜18 hrs with TNFα and TNFβ in the concentration ofexpressing 100% cytotoxicity (0.5˜2 ng/ml), and with serially 10 timesdiluted TNFR sample. Then, the cells in the 96-well plate were stainedby the staining reagent, crystal violet (Wako Pure Chemical Industries,Japan) and the activity of the cells was estimated by the degree ofabsorbance at 595 nm wavelength using a spectrophotometer (Bio-Rad,Model-550, Japan).

[0179] As shown in Table 11 represented by IC₅₀ of each TNFR/Fc fusionprotein, concatameric fusion proteins (TNFR1-TNFR1/Ig andTNFR2-TNFR2/Ig) have shown the higher inhibitory effect on thecytotoxicity induced by two kinds of TNF than simple dimeric fusionproteins (TNFR1/Ig and TNFR2/Ig). Also, as compared with the effects ofexisting simple fusion dimer and concatameric shaped TNFR/Fc fusionprotein dimer of the present invention on the inhibition of cytotoxicityof TNFα(FIG. 16) and TNFβ (FIG. 17), it more clearly appeared thatconcatameric shaped TNFR/Fc fusion protein dimers of the presentinvention remarkably inhibited the TNFα and TNFβ cytotoxicity. TABLE 11IC₅₀ of cytotoxicity inhibition IC₅₀ (ug/ml) Fusion proteins TNFαtreated TNFβ treated Simple dimer [TNFR1/Fc]₂ 63 129 [TNFR2/Fc]₂ 189 469Concatameric [TNFR1-TNFR1/Fc]₂ 9 20 dimer [TNFR2-TNFR2/Fc]₂ 15 15

EXAMPLE 7 Experiment of Suppressive Effect of Simple/Concatameric FusionDimeric CD2/Fc Fusion Protein and CTLA4/Fc Fusion Protein on theProliferation of Active Immune Cell

[0180] WT100B1S, a cell line of B lymphocyte which was made bytransfection of pyrexia patient's B lymphocyte with Ebstein-Barr viruswas incubated in RPMI 1640 supplemented with 10% FBS to use as antigenpresenting cell of T lymphocyte. After centrifuged at 2,000 rpm for 2min to precipitate, this cells were resuspended in RPMI 1640supplemented with 10% FBS to make 5.0×10⁵ cells/ml, then irradiated by3,000 rd of γ-ray.

[0181] T lymphocytes were isolated from blood of healthy adult usingFicoll-hypaque (Amersham, USA), then incubated RPMI 1640 supplementedwith 10% FBS to 2.0×10⁶ cells/ml.

[0182] To perform primary Mixed Lymphocyte Reaction (MLR), each 15 ml ofWT100B1S and T lymphocyte were mixed in 150 mm cell culture dish, andincubated for 3 days, then added by 15 ml of RPMI 1640 supplemented with10% FBS and incubated for 3 days further. After incubated for total 6days, live T lymphocytes were purified using Ficoll-hypaque (Amersham,USA) as described above, and purified T lymphocytes were stored inliquid nitrogen after freezing it by using the media comprising 45% FBS,45% RPMI 1640, and 10% DMSO.

[0183] After T lymphocytes which were reacted by primary MLR were thawedto perform secondary MLR, the cells were washed with RPMI 1640 media for2 times and made to be 3.0×10⁵ cells/ml in RPMI 1640 supplemented with10% FBS.

[0184] WT100B1S using as antigen presenting cell was newly cultured bythe method as described above, then prepared by irradiation of 3,000 rdof γ-ray and to be 7.5×10⁴ cells/ml in RPMI 1640 supplemented with 10%FBS. After 100 μl of prepared WT100B1S was added in 96-well flat bottomcell culture plate and mixed with CD2/Fc and CTLA4/Fc fusion protein atfinal concentration of 10, 1, 10⁻¹, 10⁻², 10⁻³, and 10^(−4 μ)g/ml, 100μl of primary MLR reacted T lymphocytes as above was added. Afterincubated for 2 days in 5% CO₂, 37° C. incubator, 100 μl of RPMI 1640supplemented with 10% FBS was added and incubated for 2 days further. Inthe last 6 hrs of the total 6 days culture, cells were incubated withaddition of 1.2 μCi/ml of ³H-thymidine (Amersham, USA).

[0185] At the end of culturing, supernatants were removed aftercentriftigation of 96-well plate was performed at 4° C., 110×g for 10min to precipitate T lymphocytes, and pellets were washed with 200 μl of1×PBS. Centrifugation was performed in the same condition and PBS wasremoved, then 200 μl of ice-cold trichloridic acid (TCA, Merck, USA) wasadded and mixed for 2 min, then reacted at 4° C. for 5 min to removeresidue of ³H-thymidine.

[0186] After centrifugation in the same condition as described above,supernatants were removed and T lymphocytes were fixed by incubation at4° C. for 5 min after 200 μl of ice-cold 70% ethanol was added.Supernatants were removed after centrifugation, and ³H-thymidine(Amersham, USA) residue was completely removed by treatment of 10% TCAin the same method as described above.

[0187] Cell lysis was performed by reaction with 100 μl of 2% SDS (pH8.0) and 0.5N of NaOH at 37° C. for 30 min, and T lymphocytes wereprecipitated by centrifugation at 25° C., 110×g for 10 min, and then 50μl of supernatants was transferred to 96-well sample plate (Wallac,USA). After 1.5 volume of OptiPhase SuperMix (Wallac, USA) was addedinto the supernatants and mixed for 5 min, the existence of T lymphocyteproliferation was confirmed by measurement of cpm value of ³H using 1450MicroBeta TriLux microplate liquid scintillation and luminescencecounter (Wallac, USA).

EXAMPLE 8 Experiment of Effect on Increase of Plasma Half-life ofGlycosylated Concatameric Fusion Dimeric Proteins in Mouse

[0188] The measurement of plasma half-life of glycosylated concatamericfusion dimeric proteins, [mgTNFR1-TNFR1/Fc]2, [mgTNFR2-TNFR2/Fc]2,[mgCD2-CD2/Fc]2, and [mgCTLA4-CTLA4/Fc]2 was performed by measuring theconcentration of proteins using ELISA after 5 μg of purified fusionproteins was i.p. injected into mouse (ICR, Samtako, Korea) and bloodswere extracted at regular interval for 120 hrs (5 days) as maximum. Asshown FIG. 20, FIG. 21, and FIG. 22, it could be seen that the plasmahalf-life of glycosylated concatameric fusion dimeric proteins have beenincreased in comparison of the corresponding simple fusion dimericproteins of native shape, and the increase in efficacy throughcontinuous effect could be expected.

EXAMPLE 9 Experiment of Effects of Simple/Concatameric TNFR/Fc FusionProtein Dimers on Collagen-induced Arthritis of DBA/1 Mouse

[0189] Collagen Induced Arthritis (CIA) was developed by injection with100 μg per DBA/1 mouse of type II collagen dissolved at 2 mg/mlconcentration in 0.05M acetic acid and Arthrogen-CIA adjuvant (Chondrex,USA) into tail. Boosting was performed after 3 weeks, and incompleteFreund's adjuvant (Difco, USA) was used.

[0190] Arthritis was developed 3˜4 weeks after immunization with 100 μgof type II collagen in the DBA/1 mice. Red and swollen paws of mice hadbeen observed 3˜5 days after onset, and inflammatory arthritis lastedmore than 3-4 weeks. Although inflammation was eventually alleviated,damaged joints remained rigid permanently. The degree of arthritis wasmeasured 2˜3 times per week on the basis of table 12 which representedsubjective index of arthritis severity (measure average of five mice ineach experiment). To measure the effects of simple and concatamericfusion dimeric TNFR/Fc on CIA, TNFR/Fc or PBS was i.p. injected into themice. TNFR/Fc was injected with 10 μg at every 2 days for 19˜45 daysinto 5 mice per experiments (arrows in FIG. 23). PBS was injected into 5mice as control. As shown in FIG. 7, in the case of mice injected withexisting simple dimeric shaped TNFR/Fc fusion protein, it could be seenthat the effect decreased to about 26-38% in comparison with the figuresof arthritis index in mice injected with PBS as control, but 42-55%decreased in case of concatameric shaped dimer, [TNFR1-TNFR1/Fc]₂ and[TNFR2-TNFR2/Fc]₂ were injected. Therefore, it could be shown thatconcatameric fusion dimeric TNFR/Fc fusion proteins have remarkablydecreased arthritis of mouse than existing simple fusion dimeric TNFR/Fcfusion proteins. TABLE 12 Severity score of arthritis Severity scoreCondition of disease 0 No erythema and swelling 1 Erythema and mildswelling limited to ankle and tarsal 2 Erythema and mild swelling spreadfrom ankle to tarsal 3 Erythema and mild swelling spread from ankle tometatarsal joint 4 Erythema and severe swelling expend to ankle, legs,and digits

[0191] The results as above represented that concatameric shaped dimericTNFR/Fc fusion proteins were more effective in decreasing the rate ofCIA development than existing simple dimeric fusion proteins, therefore,as use in arthritis therapy, concatameric shaped protein compositionscould be more effective therapeutics than existing protein compositions.

[0192] The concatameric proteins, concatameric fusion dimeric proteinsand their glycosylated proteins of the present invention were able toexpress increased efficacy and high stability, and to be produced withhigh yield.

[0193] Industrial Applicability

1 52 1 1335 DNA Homo sapiens CDS (1)..(1332) TNFR1-IgG 1 atg ggc ctc tccacc gtg cct gac ctg ctg ctg ccg ctg gtg ctc ctg 48 Met Gly Leu Ser ThrVal Pro Asp Leu Leu Leu Pro Leu Val Leu Leu 1 5 10 15 gag ctg ttg gtggga ata tac ccc tca ggg gtt att gga ctg gtc cct 96 Glu Leu Leu Val GlyIle Tyr Pro Ser Gly Val Ile Gly Leu Val Pro 20 25 30 cac cta ggg gac agggag aag aga gat agt gtg tgt ccc caa gga aaa 144 His Leu Gly Asp Arg GluLys Arg Asp Ser Val Cys Pro Gln Gly Lys 35 40 45 tat atc cac cct caa aataat tcg att tgc tgt acc aag tgc cac aaa 192 Tyr Ile His Pro Gln Asn AsnSer Ile Cys Cys Thr Lys Cys His Lys 50 55 60 gga acc tac ttg tac aat gactgt cca ggc ccg ggg cag gat acg gac 240 Gly Thr Tyr Leu Tyr Asn Asp CysPro Gly Pro Gly Gln Asp Thr Asp 65 70 75 80 tgc agg gag tgt gag agc ggctcc ttc acc gct tca gaa aac cac ctc 288 Cys Arg Glu Cys Glu Ser Gly SerPhe Thr Ala Ser Glu Asn His Leu 85 90 95 aga cac tgc ctc agc tgc tcc aaatgc cga aag gaa atg ggt cag gtg 336 Arg His Cys Leu Ser Cys Ser Lys CysArg Lys Glu Met Gly Gln Val 100 105 110 gag atc tct tct tgc aca gtg gaccgg gac acc gtg tgt ggc tgc agg 384 Glu Ile Ser Ser Cys Thr Val Asp ArgAsp Thr Val Cys Gly Cys Arg 115 120 125 aag aac cag tac cgg cat tat tggagt gaa aac ctt ttc cag tgc ttc 432 Lys Asn Gln Tyr Arg His Tyr Trp SerGlu Asn Leu Phe Gln Cys Phe 130 135 140 aat tgc agc ctc tgc ctc aat gggacc gtg cac ctc tcc tgc cag gag 480 Asn Cys Ser Leu Cys Leu Asn Gly ThrVal His Leu Ser Cys Gln Glu 145 150 155 160 aaa cag aac acc gtg tgc acctgc cat gca ggt ttc ttt cta aga gaa 528 Lys Gln Asn Thr Val Cys Thr CysHis Ala Gly Phe Phe Leu Arg Glu 165 170 175 aac gag tgt gtc tcc tgt agtaac tgt aag aaa agc ctg gag tgc acg 576 Asn Glu Cys Val Ser Cys Ser AsnCys Lys Lys Ser Leu Glu Cys Thr 180 185 190 aag ttg tgc cta ccc cag attgag aat gtt aag ggc act gag gac tca 624 Lys Leu Cys Leu Pro Gln Ile GluAsn Val Lys Gly Thr Glu Asp Ser 195 200 205 ggc acc aca gca gag ccc aaatct tgt gac aaa act cac aca tgc cca 672 Gly Thr Thr Ala Glu Pro Lys SerCys Asp Lys Thr His Thr Cys Pro 210 215 220 ccg tgc cca gca cct gaa ctcctg ggg gga ccg tca gtc ttc ctc ttc 720 Pro Cys Pro Ala Pro Glu Leu LeuGly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 ccc cca aaa ccc aag gacacc ctc atg atc tcc cgg acc cct gag gtc 768 Pro Pro Lys Pro Lys Asp ThrLeu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 aca tgc gtg gtg gtg gacgtg agc cac gaa gac cct gag gtc aag ttc 816 Thr Cys Val Val Val Asp ValSer His Glu Asp Pro Glu Val Lys Phe 260 265 270 aac tgg tac gtg gac ggcgtg gag gtg cat aat gcc aag aca aag ccg 864 Asn Trp Tyr Val Asp Gly ValGlu Val His Asn Ala Lys Thr Lys Pro 275 280 285 cgg gag gag cag tac aacagc acg tac cgg gtg gtc agc gtc ctc acc 912 Arg Glu Glu Gln Tyr Asn SerThr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 gtc ctg cac cag gac tggctg aat ggc aag gag tac aag tgc aag gtc 960 Val Leu His Gln Asp Trp LeuAsn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 tcc aac aaa gcc ctccca gcc ccc atc gag aaa acc atc tcc aaa gcc 1008 Ser Asn Lys Ala Leu ProAla Pro Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 aaa ggg cag ccc cgagaa cca cag gtg tac acc ctg ccc cca tcc cgg 1056 Lys Gly Gln Pro Arg GluPro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350 gat gag ctg acc aagaac cag gtc agc ctg acc tgc ctg gtc aaa ggc 1104 Asp Glu Leu Thr Lys AsnGln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 ttc tat ccc agc gacatc gcc gtg gag tgg gag agc aat ggg cag ccg 1152 Phe Tyr Pro Ser Asp IleAla Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 gag aac aac tac aagacc acg cct ccc gtg ctg gac tcc gac ggc tcc 1200 Glu Asn Asn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 tcc ttc ctc tacagc aag ctc acc gtg gac aag agc agg tgg cag cag 1248 Ser Phe Leu Tyr SerLys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415 ggg aac gtc ttctca tgc tcc gtg atg cat gag gct ctg cac aac cac 1296 Gly Asn Val Phe SerCys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 tac acg cag aagagc ctc tcc ctg tct ccg ggt aaa tga 1335 Tyr Thr Gln Lys Ser Leu Ser LeuSer Pro Gly Lys 435 440 2 444 PRT Homo sapiens 2 Met Gly Leu Ser Thr ValPro Asp Leu Leu Leu Pro Leu Val Leu Leu 1 5 10 15 Glu Leu Leu Val GlyIle Tyr Pro Ser Gly Val Ile Gly Leu Val Pro 20 25 30 His Leu Gly Asp ArgGlu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys 35 40 45 Tyr Ile His Pro GlnAsn Asn Ser Ile Cys Cys Thr Lys Cys His Lys 50 55 60 Gly Thr Tyr Leu TyrAsn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp 65 70 75 80 Cys Arg Glu CysGlu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu 85 90 95 Arg His Cys LeuSer Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val 100 105 110 Glu Ile SerSer Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg 115 120 125 Lys AsnGln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe 130 135 140 AsnCys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu 145 150 155160 Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu 165170 175 Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr180 185 190 Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu AspSer 195 200 205 Gly Thr Thr Ala Glu Pro Lys Ser Cys Asp Lys Thr His ThrCys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser ValPhe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile SerArg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser His GluAsp Pro Glu Val Lys Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Tyr Asn Ser ThrTyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp LeuAsn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Ala LeuPro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln ProArg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350 Asp Glu LeuThr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe TyrPro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 GluAsn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395400 Ser Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 31473 DNA Homo sapiens CDS (1)..(1470) TNFR2-IgG 3 atg gcg ccc gtc gccgtc tgg gcc gcg ctg gcc gtc gga ctg gag ctc 48 Met Ala Pro Val Ala ValTrp Ala Ala Leu Ala Val Gly Leu Glu Leu 1 5 10 15 tgg gct gcg gcg cacgcc ttg ccc gcc cag gtg gca ttt aca ccc tac 96 Trp Ala Ala Ala His AlaLeu Pro Ala Gln Val Ala Phe Thr Pro Tyr 20 25 30 gcc ccg gag ccc ggg agcaca tgc cgg ctc aga gaa tac tat gac cag 144 Ala Pro Glu Pro Gly Ser ThrCys Arg Leu Arg Glu Tyr Tyr Asp Gln 35 40 45 aca gct cag atg tgc tgc agcaaa tgc tcg ccg ggc caa cat gca aaa 192 Thr Ala Gln Met Cys Cys Ser LysCys Ser Pro Gly Gln His Ala Lys 50 55 60 gtc ttc tgt acc aag acc tcg gacacc gtg tgt gac tcc tgt gag gac 240 Val Phe Cys Thr Lys Thr Ser Asp ThrVal Cys Asp Ser Cys Glu Asp 65 70 75 80 agc aca tac acc cag ctc tgg aactgg gtt ccc gag tgc ttg agc tgt 288 Ser Thr Tyr Thr Gln Leu Trp Asn TrpVal Pro Glu Cys Leu Ser Cys 85 90 95 ggc tcc cgc tgt agc tct gac cag gtggaa act caa gcc tgc act cgg 336 Gly Ser Arg Cys Ser Ser Asp Gln Val GluThr Gln Ala Cys Thr Arg 100 105 110 gaa cag aac cgc atc tgc acc tgc aggccc ggc tgg tac tgc gcg ctg 384 Glu Gln Asn Arg Ile Cys Thr Cys Arg ProGly Trp Tyr Cys Ala Leu 115 120 125 agc aag cag gag ggg tgc cgg ctg tgcgcg ccg ctg cgc aag tgc cgc 432 Ser Lys Gln Glu Gly Cys Arg Leu Cys AlaPro Leu Arg Lys Cys Arg 130 135 140 ccg ggc ttc ggc gtg gcc aga cca ggaact gaa aca tca gac gtg gtg 480 Pro Gly Phe Gly Val Ala Arg Pro Gly ThrGlu Thr Ser Asp Val Val 145 150 155 160 tgc aag ccc tgt gcc ccg ggg acgttc tcc aac acg act tca tcc acg 528 Cys Lys Pro Cys Ala Pro Gly Thr PheSer Asn Thr Thr Ser Ser Thr 165 170 175 gat att tgc agg ccc cac cag atctgt aac gtg gtg gcc atc cct ggg 576 Asp Ile Cys Arg Pro His Gln Ile CysAsn Val Val Ala Ile Pro Gly 180 185 190 aat gca agc atg gat gca gtc tgcacg tcc acg tcc ccc acc cgg agt 624 Asn Ala Ser Met Asp Ala Val Cys ThrSer Thr Ser Pro Thr Arg Ser 195 200 205 atg gcc cca ggg gca gta cac ttaccc cag cca gtg tcc aca cga tcc 672 Met Ala Pro Gly Ala Val His Leu ProGln Pro Val Ser Thr Arg Ser 210 215 220 caa cac acg cag cca act cca gaaccc agc act gct cca agc acc tcc 720 Gln His Thr Gln Pro Thr Pro Glu ProSer Thr Ala Pro Ser Thr Ser 225 230 235 240 ttc ctg ctc cca atg ggc cccagc ccc cca gct gaa ggg agc act ggc 768 Phe Leu Leu Pro Met Gly Pro SerPro Pro Ala Glu Gly Ser Thr Gly 245 250 255 gac gca gag ccc aaa tct tgtgac aaa act cac aca tgc cca ccg tgc 816 Asp Ala Glu Pro Lys Ser Cys AspLys Thr His Thr Cys Pro Pro Cys 260 265 270 cca gca cct gaa ctc ctg ggggga ccg tca gtc ttc ctc ttc ccc cca 864 Pro Ala Pro Glu Leu Leu Gly GlyPro Ser Val Phe Leu Phe Pro Pro 275 280 285 aaa ccc aag gac acc ctc atgatc tcc cgg acc cct gag gtc aca tgc 912 Lys Pro Lys Asp Thr Leu Met IleSer Arg Thr Pro Glu Val Thr Cys 290 295 300 gtg gtg gtg gac gtg agc cacgaa gac cct gag gtc aag ttc aac tgg 960 Val Val Val Asp Val Ser His GluAsp Pro Glu Val Lys Phe Asn Trp 305 310 315 320 tac gtg gac ggc gtg gaggtg cat aat gcc aag aca aag ccg cgg gag 1008 Tyr Val Asp Gly Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu 325 330 335 gag cag tac aac agc acgtac cgg gtg gtc agc gtc ctc acc gtc ctg 1056 Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val Leu 340 345 350 cac cag gac tgg ctg aatggc aag gag tac aag tgc aag gtc tcc aac 1104 His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn 355 360 365 aaa gcc ctc cca gcc cccatc gag aaa acc atc tcc aaa gcc aaa ggg 1152 Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly 370 375 380 cag ccc cga gaa cca caggtg tac acc ctg ccc cca tcc cgg gat gag 1200 Gln Pro Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu 385 390 395 400 ctg acc aag aac caggtc agc ctg acc tgc ctg gtc aaa ggc ttc tat 1248 Leu Thr Lys Asn Gln ValSer Leu Thr Cys Leu Val Lys Gly Phe Tyr 405 410 415 ccc agc gac atc gccgtg gag tgg gag agc aat ggg cag ccg gag aac 1296 Pro Ser Asp Ile Ala ValGlu Trp Glu Ser Asn Gly Gln Pro Glu Asn 420 425 430 aac tac aag acc acgcct ccc gtg ctg gac tcc gac ggc tcc tcc ttc 1344 Asn Tyr Lys Thr Thr ProPro Val Leu Asp Ser Asp Gly Ser Ser Phe 435 440 445 ctc tac agc aag ctcacc gtg gac aag agc agg tgg cag cag ggg aac 1392 Leu Tyr Ser Lys Leu ThrVal Asp Lys Ser Arg Trp Gln Gln Gly Asn 450 455 460 gtc ttc tca tgc tccgtg atg cat gag gct ctg cac aac cac tac acg 1440 Val Phe Ser Cys Ser ValMet His Glu Ala Leu His Asn His Tyr Thr 465 470 475 480 cag aag agc ctctcc ctg tct ccg ggt aaa tga 1473 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys485 490 4 490 PRT Homo sapiens 4 Met Ala Pro Val Ala Val Trp Ala Ala LeuAla Val Gly Leu Glu Leu 1 5 10 15 Trp Ala Ala Ala His Ala Leu Pro AlaGln Val Ala Phe Thr Pro Tyr 20 25 30 Ala Pro Glu Pro Gly Ser Thr Cys ArgLeu Arg Glu Tyr Tyr Asp Gln 35 40 45 Thr Ala Gln Met Cys Cys Ser Lys CysSer Pro Gly Gln His Ala Lys 50 55 60 Val Phe Cys Thr Lys Thr Ser Asp ThrVal Cys Asp Ser Cys Glu Asp 65 70 75 80 Ser Thr Tyr Thr Gln Leu Trp AsnTrp Val Pro Glu Cys Leu Ser Cys 85 90 95 Gly Ser Arg Cys Ser Ser Asp GlnVal Glu Thr Gln Ala Cys Thr Arg 100 105 110 Glu Gln Asn Arg Ile Cys ThrCys Arg Pro Gly Trp Tyr Cys Ala Leu 115 120 125 Ser Lys Gln Glu Gly CysArg Leu Cys Ala Pro Leu Arg Lys Cys Arg 130 135 140 Pro Gly Phe Gly ValAla Arg Pro Gly Thr Glu Thr Ser Asp Val Val 145 150 155 160 Cys Lys ProCys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr 165 170 175 Asp IleCys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly 180 185 190 AsnAla Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser 195 200 205Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser 210 215220 Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser 225230 235 240 Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser ThrGly 245 250 255 Asp Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys ProPro Cys 260 265 270 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe LeuPhe Pro Pro 275 280 285 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr ProGlu Val Thr Cys 290 295 300 Val Val Val Asp Val Ser His Glu Asp Pro GluVal Lys Phe Asn Trp 305 310 315 320 Tyr Val Asp Gly Val Glu Val His AsnAla Lys Thr Lys Pro Arg Glu 325 330 335 Glu Gln Tyr Asn Ser Thr Tyr ArgVal Val Ser Val Leu Thr Val Leu 340 345 350 His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn 355 360 365 Lys Ala Leu Pro Ala ProIle Glu Lys Thr Ile Ser Lys Ala Lys Gly 370 375 380 Gln Pro Arg Glu ProGln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 385 390 395 400 Leu Thr LysAsn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 405 410 415 Pro SerAsp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 420 425 430 AsnTyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Ser Phe 435 440 445Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 450 455460 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 465470 475 480 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 490 5 1887 DNAHomo sapiens CDS (1)..(1884) TNFR1-TNFR1-IgG 5 atg ggc ctc tcc acc gtgcct gac ctg ctg ctg ccg ctg gtg ctc ctg 48 Met Gly Leu Ser Thr Val ProAsp Leu Leu Leu Pro Leu Val Leu Leu 1 5 10 15 gag ctg ttg gtg gga atatac ccc tca ggg gtt att gga ctg gtc cct 96 Glu Leu Leu Val Gly Ile TyrPro Ser Gly Val Ile Gly Leu Val Pro 20 25 30 cac cta ggg gac agg gag aagaga gat agt gtg tgt ccc caa gga aaa 144 His Leu Gly Asp Arg Glu Lys ArgAsp Ser Val Cys Pro Gln Gly Lys 35 40 45 tat atc cac cct caa aat aat tcgatt tgc tgt acc aag tgc cac aaa 192 Tyr Ile His Pro Gln Asn Asn Ser IleCys Cys Thr Lys Cys His Lys 50 55 60 gga acc tac ttg tac aat gac tgt ccaggc ccg ggg cag gat acg gac 240 Gly Thr Tyr Leu Tyr Asn Asp Cys Pro GlyPro Gly Gln Asp Thr Asp 65 70 75 80 tgc agg gag tgt gag agc ggc tcc ttcacc gct tca gaa aac cac ctc 288 Cys Arg Glu Cys Glu Ser Gly Ser Phe ThrAla Ser Glu Asn His Leu 85 90 95 aga cac tgc ctc agc tgc tcc aaa tgc cgaaag gaa atg ggt cag gtg 336 Arg His Cys Leu Ser Cys Ser Lys Cys Arg LysGlu Met Gly Gln Val 100 105 110 gag atc tct tct tgc aca gtg gac cgg gacacc gtg tgt ggc tgc agg 384 Glu Ile Ser Ser Cys Thr Val Asp Arg Asp ThrVal Cys Gly Cys Arg 115 120 125 aag aac cag tac cgg cat tat tgg agt gaaaac ctt ttc cag tgc ttc 432 Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu AsnLeu Phe Gln Cys Phe 130 135 140 aat tgc agc ctc tgc ctc aat ggg acc gtgcac ctc tcc tgc cag gag 480 Asn Cys Ser Leu Cys Leu Asn Gly Thr Val HisLeu Ser Cys Gln Glu 145 150 155 160 aaa cag aac acc gtg tgc acc tgc catgca ggt ttc ttt cta aga gaa 528 Lys Gln Asn Thr Val Cys Thr Cys His AlaGly Phe Phe Leu Arg Glu 165 170 175 aac gag tgt gtc tcc tgt agt aac tgtaag aaa agc ctg gag tgc acg 576 Asn Glu Cys Val Ser Cys Ser Asn Cys LysLys Ser Leu Glu Cys Thr 180 185 190 aag ttg tgc cta ccc cag att gag aatgtt aag ggc act gag gac gga 624 Lys Leu Cys Leu Pro Gln Ile Glu Asn ValLys Gly Thr Glu Asp Gly 195 200 205 tcc ggg aac att tca ctg gtc cct caccta ggg gac agg gag aag aga 672 Ser Gly Asn Ile Ser Leu Val Pro His LeuGly Asp Arg Glu Lys Arg 210 215 220 gat agt gtg tgt ccc caa gga aaa tatatc cac cct caa aat aat tcg 720 Asp Ser Val Cys Pro Gln Gly Lys Tyr IleHis Pro Gln Asn Asn Ser 225 230 235 240 att tgc tgt acc aag tgc cac aaagga acc tac ttg tac aat gac tgt 768 Ile Cys Cys Thr Lys Cys His Lys GlyThr Tyr Leu Tyr Asn Asp Cys 245 250 255 cca ggc ccg ggg cag gat acg gactgc agg gag tgt gag agc ggc tcc 816 Pro Gly Pro Gly Gln Asp Thr Asp CysArg Glu Cys Glu Ser Gly Ser 260 265 270 ttc acc gct tca gaa aac cac ctcaga cac tgc ctc agc tgc tcc aaa 864 Phe Thr Ala Ser Glu Asn His Leu ArgHis Cys Leu Ser Cys Ser Lys 275 280 285 tgc cga aag gaa atg ggt cag gtggag atc tct tct tgc aca gtg gac 912 Cys Arg Lys Glu Met Gly Gln Val GluIle Ser Ser Cys Thr Val Asp 290 295 300 cgg gac acc gtg tgt ggc tgc aggaag aac cag tac cgg cat tat tgg 960 Arg Asp Thr Val Cys Gly Cys Arg LysAsn Gln Tyr Arg His Tyr Trp 305 310 315 320 agt gaa aac ctt ttc cag tgcttc aat tgc agc ctc tgc ctc aat ggg 1008 Ser Glu Asn Leu Phe Gln Cys PheAsn Cys Ser Leu Cys Leu Asn Gly 325 330 335 acc gtg cac ctc tcc tgc caggag aaa cag aac acc gtg tgc acc tgc 1056 Thr Val His Leu Ser Cys Gln GluLys Gln Asn Thr Val Cys Thr Cys 340 345 350 cat gca ggt ttc ttt cta agagaa aac gag tgt gtc tcc tgt agt aac 1104 His Ala Gly Phe Phe Leu Arg GluAsn Glu Cys Val Ser Cys Ser Asn 355 360 365 tgt aag aaa agc ctg gag tgcacg aag ttg tgc cta ccc cag att gag 1152 Cys Lys Lys Ser Leu Glu Cys ThrLys Leu Cys Leu Pro Gln Ile Glu 370 375 380 aat gtt aag ggc act gag gactca ggc acc aca gca gag ccc aaa tct 1200 Asn Val Lys Gly Thr Glu Asp SerGly Thr Thr Ala Glu Pro Lys Ser 385 390 395 400 tgt gac aaa act cac acatgc cca ccg tgc cca gca cct gaa ctc ctg 1248 Cys Asp Lys Thr His Thr CysPro Pro Cys Pro Ala Pro Glu Leu Leu 405 410 415 ggg gga ccg tca gtc ttcctc ttc ccc cca aaa ccc aag gac acc ctc 1296 Gly Gly Pro Ser Val Phe LeuPhe Pro Pro Lys Pro Lys Asp Thr Leu 420 425 430 atg atc tcc cgg acc cctgag gtc aca tgc gtg gtg gtg gac gtg agc 1344 Met Ile Ser Arg Thr Pro GluVal Thr Cys Val Val Val Asp Val Ser 435 440 445 cac gaa gac cct gag gtcaag ttc aac tgg tac gtg gac ggc gtg gag 1392 His Glu Asp Pro Glu Val LysPhe Asn Trp Tyr Val Asp Gly Val Glu 450 455 460 gtg cat aat gcc aag acaaag ccg cgg gag gag cag tac aac agc acg 1440 Val His Asn Ala Lys Thr LysPro Arg Glu Glu Gln Tyr Asn Ser Thr 465 470 475 480 tac cgg gtg gtc agcgtc ctc acc gtc ctg cac cag gac tgg ctg aat 1488 Tyr Arg Val Val Ser ValLeu Thr Val Leu His Gln Asp Trp Leu Asn 485 490 495 ggc aag gag tac aagtgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 1536 Gly Lys Glu Tyr Lys CysLys Val Ser Asn Lys Ala Leu Pro Ala Pro 500 505 510 atc gag aaa acc atctcc aaa gcc aaa ggg cag ccc cga gaa cca cag 1584 Ile Glu Lys Thr Ile SerLys Ala Lys Gly Gln Pro Arg Glu Pro Gln 515 520 525 gtg tac acc ctg ccccca tcc cgg gat gag ctg acc aag aac cag gtc 1632 Val Tyr Thr Leu Pro ProSer Arg Asp Glu Leu Thr Lys Asn Gln Val 530 535 540 agc ctg acc tgc ctggtc aaa ggc ttc tat ccc agc gac atc gcc gtg 1680 Ser Leu Thr Cys Leu ValLys Gly Phe Tyr Pro Ser Asp Ile Ala Val 545 550 555 560 gag tgg gag agcaat ggg cag ccg gag aac aac tac aag acc acg cct 1728 Glu Trp Glu Ser AsnGly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 565 570 575 ccc gtg ctg gactcc gac ggc tcc tcc ttc ctc tac agc aag ctc acc 1776 Pro Val Leu Asp SerAsp Gly Ser Ser Phe Leu Tyr Ser Lys Leu Thr 580 585 590 gtg gac aag agcagg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg 1824 Val Asp Lys Ser ArgTrp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 595 600 605 atg cat gag gctctg cac aac cac tac acg cag aag agc ctc tcc ctg 1872 Met His Glu Ala LeuHis Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 610 615 620 tct ccg ggt aaatga 1887 Ser Pro Gly Lys 625 6 628 PRT Homo sapiens 6 Met Gly Leu SerThr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu 1 5 10 15 Glu Leu LeuVal Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro 20 25 30 His Leu GlyAsp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys 35 40 45 Tyr Ile HisPro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys 50 55 60 Gly Thr TyrLeu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp 65 70 75 80 Cys ArgGlu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu 85 90 95 Arg HisCys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val 100 105 110 GluIle Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg 115 120 125Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe 130 135140 Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu 145150 155 160 Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu ArgGlu 165 170 175 Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu GluCys Thr 180 185 190 Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly ThrGlu Asp Gly 195 200 205 Ser Gly Asn Ile Ser Leu Val Pro His Leu Gly AspArg Glu Lys Arg 210 215 220 Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile HisPro Gln Asn Asn Ser 225 230 235 240 Ile Cys Cys Thr Lys Cys His Lys GlyThr Tyr Leu Tyr Asn Asp Cys 245 250 255 Pro Gly Pro Gly Gln Asp Thr AspCys Arg Glu Cys Glu Ser Gly Ser 260 265 270 Phe Thr Ala Ser Glu Asn HisLeu Arg His Cys Leu Ser Cys Ser Lys 275 280 285 Cys Arg Lys Glu Met GlyGln Val Glu Ile Ser Ser Cys Thr Val Asp 290 295 300 Arg Asp Thr Val CysGly Cys Arg Lys Asn Gln Tyr Arg His Tyr Trp 305 310 315 320 Ser Glu AsnLeu Phe Gln Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly 325 330 335 Thr ValHis Leu Ser Cys Gln Glu Lys Gln Asn Thr Val Cys Thr Cys 340 345 350 HisAla Gly Phe Phe Leu Arg Glu Asn Glu Cys Val Ser Cys Ser Asn 355 360 365Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu Pro Gln Ile Glu 370 375380 Asn Val Lys Gly Thr Glu Asp Ser Gly Thr Thr Ala Glu Pro Lys Ser 385390 395 400 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu LeuLeu 405 410 415 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys AspThr Leu 420 425 430 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val ValAsp Val Ser 435 440 445 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr ValAsp Gly Val Glu 450 455 460 Val His Asn Ala Lys Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr 465 470 475 480 Tyr Arg Val Val Ser Val Leu Thr ValLeu His Gln Asp Trp Leu Asn 485 490 495 Gly Lys Glu Tyr Lys Cys Lys ValSer Asn Lys Ala Leu Pro Ala Pro 500 505 510 Ile Glu Lys Thr Ile Ser LysAla Lys Gly Gln Pro Arg Glu Pro Gln 515 520 525 Val Tyr Thr Leu Pro ProSer Arg Asp Glu Leu Thr Lys Asn Gln Val 530 535 540 Ser Leu Thr Cys LeuVal Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 545 550 555 560 Glu Trp GluSer Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 565 570 575 Pro ValLeu Asp Ser Asp Gly Ser Ser Phe Leu Tyr Ser Lys Leu Thr 580 585 590 ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 595 600 605Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 610 615620 Ser Pro Gly Lys 625 7 2163 DNA Homo sapiens CDS (1)..(2160)TNFR2-TNFR2-IgG 7 atg gcg ccc gtc gcc gtc tgg gcc gcg ctg gcc gtc ggactg gag ctc 48 Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly LeuGlu Leu 1 5 10 15 tgg gct gcg gcg cac gcc ttg ccc gcc cag gtg gca tttaca ccc tac 96 Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe ThrPro Tyr 20 25 30 gcc ccg gag ccc ggg agc aca tgc cgg ctc aga gaa tac tatgac cag 144 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr AspGln 35 40 45 aca gct cag atg tgc tgc agc aaa tgc tcg ccg ggc caa cat gcaaaa 192 Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys50 55 60 gtc ttc tgt acc aag acc tcg gac acc gtg tgt gac tcc tgt gag gac240 Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp 6570 75 80 agc aca tac acc cag ctc tgg aac tgg gtt ccc gag tgc ttg agc tgt288 Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys 8590 95 ggc tcc cgc tgt agc tct gac cag gtg gaa act caa gcc tgc act cgg336 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg 100105 110 gaa cag aac cgc atc tgc acc tgc agg ccc ggc tgg tac tgc gcg ctg384 Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 115120 125 agc aag cag gag ggg tgc cgg ctg tgc gcg ccg ctg cgc aag tgc cgc432 Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg 130135 140 ccg ggc ttc ggc gtg gcc aga cca gga act gaa aca tca gac gtg gtg480 Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val 145150 155 160 tgc aag ccc tgt gcc ccg ggg acg ttc tcc aac acg act tca tccacg 528 Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr165 170 175 gat att tgc agg ccc cac cag atc tgt aac gtg gtg gcc atc cctggg 576 Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly180 185 190 aat gca agc atg gat gca gtc tgc acg tcc acg tcc ccc acc cggagt 624 Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser195 200 205 atg gcc cca ggg gca gta cac tta ccc cag cca gtg tcc aca cgatcc 672 Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser210 215 220 caa cac acg cag cca act cca gaa ccc agc act gct cca agc acctcc 720 Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser225 230 235 240 ttc ctg ctc cca atg ggc ccc agc ccc cca gct gaa ggg agcgga tcc 768 Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser GlySer 245 250 255 aac gca act aca ccc tac gcc ccg gag ccc ggg agc aca tgccgg ctc 816 Asn Ala Thr Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys ArgLeu 260 265 270 aga gaa tac tat gac cag aca gct cag atg tgc tgc agc aaatgc tcg 864 Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys Ser Lys CysSer 275 280 285 ccg ggc caa cat gca aaa gtc ttc tgt acc aag acc tcg gacacc gtg 912 Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr Ser Asp ThrVal 290 295 300 tgt gac tcc tgt gag gac agc aca tac acc cag ctc tgg aactgg gtt 960 Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn TrpVal 305 310 315 320 ccc gag tgc ttg agc tgt ggc tcc cgc tgt agc tct gaccag gtg gaa 1008 Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp GlnVal Glu 325 330 335 act caa gcc tgc act cgg gaa cag aac cgc atc tgc acctgc agg ccc 1056 Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr CysArg Pro 340 345 350 ggc tgg tac tgc gcg ctg agc aag cag gag ggg tgc cggctg tgc gcg 1104 Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg LeuCys Ala 355 360 365 ccg ctg cgc aag tgc cgc ccg ggc ttc ggc gtg gcc agacca gga act 1152 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg ProGly Thr 370 375 380 gaa aca tca gac gtg gtg tgc aag ccc tgt gcc ccg gggacg ttc tcc 1200 Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro Gly ThrPhe Ser 385 390 395 400 aac acg act tca tcc acg gat att tgc agg ccc caccag atc tgt aac 1248 Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His GlnIle Cys Asn 405 410 415 gtg gtg gcc atc cct ggg aat gca agc atg gat gcagtc tgc acg tcc 1296 Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala ValCys Thr Ser 420 425 430 acg tcc ccc acc cgg agt atg gcc cca ggg gca gtacac tta ccc cag 1344 Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val HisLeu Pro Gln 435 440 445 cca gtg tcc aca cga tcc caa cac acg cag cca actcca gaa ccc agc 1392 Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr ProGlu Pro Ser 450 455 460 act gct cca agc acc tcc ttc ctg ctc cca atg ggcccc agc ccc cca 1440 Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly ProSer Pro Pro 465 470 475 480 gct gaa ggg agc act ggc gac gca gag ccc aaatct tgt gac aaa act 1488 Ala Glu Gly Ser Thr Gly Asp Ala Glu Pro Lys SerCys Asp Lys Thr 485 490 495 cac aca tgc cca ccg tgc cca gca cct gaa ctcctg ggg gga ccg tca 1536 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu LeuGly Gly Pro Ser 500 505 510 gtc ttc ctc ttc ccc cca aaa ccc aag gac accctc atg atc tcc cgg 1584 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr LeuMet Ile Ser Arg 515 520 525 acc cct gag gtc aca tgc gtg gtg gtg gac gtgagc cac gaa gac cct 1632 Thr Pro Glu Val Thr Cys Val Val Val Asp Val SerHis Glu Asp Pro 530 535 540 gag gtc aag ttc aac tgg tac gtg gac ggc gtggag gtg cat aat gcc 1680 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val GluVal His Asn Ala 545 550 555 560 aag aca aag ccg cgg gag gag cag tac aacagc acg tac cgg gtg gtc 1728 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn SerThr Tyr Arg Val Val 565 570 575 agc gtc ctc acc gtc ctg cac cag gac tggctg aat ggc aag gag tac 1776 Ser Val Leu Thr Val Leu His Gln Asp Trp LeuAsn Gly Lys Glu Tyr 580 585 590 aag tgc aag gtc tcc aac aaa gcc ctc ccagcc ccc atc gag aaa acc 1824 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro AlaPro Ile Glu Lys Thr 595 600 605 atc tcc aaa gcc aaa ggg cag ccc cga gaacca cag gtg tac acc ctg 1872 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu ProGln Val Tyr Thr Leu 610 615 620 ccc cca tcc cgg gat gag ctg acc aag aaccag gtc agc ctg acc tgc 1920 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn GlnVal Ser Leu Thr Cys 625 630 635 640 ctg gtc aaa ggc ttc tat ccc agc gacatc gcc gtg gag tgg gag agc 1968 Leu Val Lys Gly Phe Tyr Pro Ser Asp IleAla Val Glu Trp Glu Ser 645 650 655 aat ggg cag ccg gag aac aac tac aagacc acg cct ccc gtg ctg gac 2016 Asn Gly Gln Pro Glu Asn Asn Tyr Lys ThrThr Pro Pro Val Leu Asp 660 665 670 tcc gac ggc tcc tcc ttc ctc tac agcaag ctc acc gtg gac aag agc 2064 Ser Asp Gly Ser Ser Phe Leu Tyr Ser LysLeu Thr Val Asp Lys Ser 675 680 685 agg tgg cag cag ggg aac gtc ttc tcatgc tcc gtg atg cat gag gct 2112 Arg Trp Gln Gln Gly Asn Val Phe Ser CysSer Val Met His Glu Ala 690 695 700 ctg cac aac cac tac acg cag aag agcctc tcc ctg tct ccg ggt aaa 2160 Leu His Asn His Tyr Thr Gln Lys Ser LeuSer Leu Ser Pro Gly Lys 705 710 715 720 tga 2163 8 720 PRT Homo sapiens8 Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu 1 5 1015 Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr 20 2530 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln 35 4045 Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys 50 5560 Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp 65 7075 80 Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys 8590 95 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg100 105 110 Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys AlaLeu 115 120 125 Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg LysCys Arg 130 135 140 Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr SerAsp Val Val 145 150 155 160 Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser AsnThr Thr Ser Ser Thr 165 170 175 Asp Ile Cys Arg Pro His Gln Ile Cys AsnVal Val Ala Ile Pro Gly 180 185 190 Asn Ala Ser Met Asp Ala Val Cys ThrSer Thr Ser Pro Thr Arg Ser 195 200 205 Met Ala Pro Gly Ala Val His LeuPro Gln Pro Val Ser Thr Arg Ser 210 215 220 Gln His Thr Gln Pro Thr ProGlu Pro Ser Thr Ala Pro Ser Thr Ser 225 230 235 240 Phe Leu Leu Pro MetGly Pro Ser Pro Pro Ala Glu Gly Ser Gly Ser 245 250 255 Asn Ala Thr ThrPro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu 260 265 270 Arg Glu TyrTyr Asp Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Ser 275 280 285 Pro GlyGln His Ala Lys Val Phe Cys Thr Lys Thr Ser Asp Thr Val 290 295 300 CysAsp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val 305 310 315320 Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu 325330 335 Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro340 345 350 Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu CysAla 355 360 365 Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg ProGly Thr 370 375 380 Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro GlyThr Phe Ser 385 390 395 400 Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg ProHis Gln Ile Cys Asn 405 410 415 Val Val Ala Ile Pro Gly Asn Ala Ser MetAsp Ala Val Cys Thr Ser 420 425 430 Thr Ser Pro Thr Arg Ser Met Ala ProGly Ala Val His Leu Pro Gln 435 440 445 Pro Val Ser Thr Arg Ser Gln HisThr Gln Pro Thr Pro Glu Pro Ser 450 455 460 Thr Ala Pro Ser Thr Ser PheLeu Leu Pro Met Gly Pro Ser Pro Pro 465 470 475 480 Ala Glu Gly Ser ThrGly Asp Ala Glu Pro Lys Ser Cys Asp Lys Thr 485 490 495 His Thr Cys ProPro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 500 505 510 Val Phe LeuPhe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 515 520 525 Thr ProGlu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 530 535 540 GluVal Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 545 550 555560 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 565570 575 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr580 585 590 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu LysThr 595 600 605 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val TyrThr Leu 610 615 620 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerLeu Thr Cys 625 630 635 640 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile AlaVal Glu Trp Glu Ser 645 650 655 Asn Gly Gln Pro Glu Asn Asn Tyr Lys ThrThr Pro Pro Val Leu Asp 660 665 670 Ser Asp Gly Ser Ser Phe Leu Tyr SerLys Leu Thr Val Asp Lys Ser 675 680 685 Arg Trp Gln Gln Gly Asn Val PheSer Cys Ser Val Met His Glu Ala 690 695 700 Leu His Asn His Tyr Thr GlnLys Ser Leu Ser Leu Ser Pro Gly Lys 705 710 715 720 9 1827 DNA Homosapiens CDS (1)..(1824) mgTNFR1-TNFR1-IgG 9 atg ggc ctc tcc acc gtg cctgac ctg ctg ctg ccg ctg gtg ctc ctg 48 Met Gly Leu Ser Thr Val Pro AspLeu Leu Leu Pro Leu Val Leu Leu 1 5 10 15 gag ctg ttg gtg gga ata tacccc tca ggg gtt att gga ctg gtc cct 96 Glu Leu Leu Val Gly Ile Tyr ProSer Gly Val Ile Gly Leu Val Pro 20 25 30 cac cta ggg gac agg gag aag agagat agt gtg tgt ccc caa gga aaa 144 His Leu Gly Asp Arg Glu Lys Arg AspSer Val Cys Pro Gln Gly Lys 35 40 45 tat atc cac cct caa aat aat tcg atttgc tgt acc aag tgc cac aaa 192 Tyr Ile His Pro Gln Asn Asn Ser Ile CysCys Thr Lys Cys His Lys 50 55 60 gga acc tac ttg tac aat gac tgt cca ggcccg ggg cag gat acg gac 240 Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly ProGly Gln Asp Thr Asp 65 70 75 80 tgc agg gag tgt gag agc ggc tcc ttc accgct tca gaa aac cac ctc 288 Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr AlaSer Glu Asn His Leu 85 90 95 aga cac tgc ctc agc tgc tcc aaa tgc cga aaggaa atg ggt cag gtg 336 Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys GluMet Gly Gln Val 100 105 110 gag atc tct tct tgc aca gtg gac cgg gac accgtg tgt ggc tgc agg 384 Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr ValCys Gly Cys Arg 115 120 125 aag aac cag tac cgg cat tat tgg agt gaa aacctt ttc cag tgc ttc 432 Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn LeuPhe Gln Cys Phe 130 135 140 aat tgc agc ctc tgc ctc aat ggg acc gtg cacctc tcc tgc cag gag 480 Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His LeuSer Cys Gln Glu 145 150 155 160 aaa cag aac acc gtg tgc acc tgc cat gcaggt ttc ttt cta aga gaa 528 Lys Gln Asn Thr Val Cys Thr Cys His Ala GlyPhe Phe Leu Arg Glu 165 170 175 aac gag tgt gtc tcc tgt agt aac tgt aagaaa agc aac gag acc aac 576 Asn Glu Cys Val Ser Cys Ser Asn Cys Lys LysSer Asn Glu Thr Asn 180 185 190 aag acc tgc cta cac aac ggg tcc agg gagaag aac gat agt gtg tgt 624 Lys Thr Cys Leu His Asn Gly Ser Arg Glu LysAsn Asp Ser Val Cys 195 200 205 ccc caa gga aaa tat atc cac cct caa aataat tcg att tgc tgt acc 672 Pro Gln Gly Lys Tyr Ile His Pro Gln Asn AsnSer Ile Cys Cys Thr 210 215 220 aag tgc cac aaa gga acc tac ttg tac aatgac tgt cca ggc ccg ggg 720 Lys Cys His Lys Gly Thr Tyr Leu Tyr Asn AspCys Pro Gly Pro Gly 225 230 235 240 cag gat acg gac tgc agg gag tgt gagagc ggc tcc ttc acc gct tca 768 Gln Asp Thr Asp Cys Arg Glu Cys Glu SerGly Ser Phe Thr Ala Ser 245 250 255 gaa aac cac ctc aga cac tgc ctc agctgc tcc aaa tgc cga aag gaa 816 Glu Asn His Leu Arg His Cys Leu Ser CysSer Lys Cys Arg Lys Glu 260 265 270 atg ggt cag gtg gag atc tct tct tgcaca gtg gac cgg gac acc gtg 864 Met Gly Gln Val Glu Ile Ser Ser Cys ThrVal Asp Arg Asp Thr Val 275 280 285 tgt ggc tgc agg aag aac cag tac cggcat tat tgg agt gaa aac ctt 912 Cys Gly Cys Arg Lys Asn Gln Tyr Arg HisTyr Trp Ser Glu Asn Leu 290 295 300 ttc cag tgc ttc aat tgc agc ctc tgcctc aat ggg acc gtg cac ctc 960 Phe Gln Cys Phe Asn Cys Ser Leu Cys LeuAsn Gly Thr Val His Leu 305 310 315 320 tcc tgc cag gag aaa cag aac accgtg tgc acc tgc cat gca ggt ttc 1008 Ser Cys Gln Glu Lys Gln Asn Thr ValCys Thr Cys His Ala Gly Phe 325 330 335 ttt cta aga gaa aac gag tgt gtctcc tgt agt aac tgt aag aaa agc 1056 Phe Leu Arg Glu Asn Glu Cys Val SerCys Ser Asn Cys Lys Lys Ser 340 345 350 ctg gag tgc acg aag ttg tgc ctaccc cag att gag aat gtt aag ggc 1104 Leu Glu Cys Thr Lys Leu Cys Leu ProGln Ile Glu Asn Val Lys Gly 355 360 365 act gag gac tca ggc acc aca gcagag ccc aaa tct tgt gac aaa act 1152 Thr Glu Asp Ser Gly Thr Thr Ala GluPro Lys Ser Cys Asp Lys Thr 370 375 380 cac aca tgc cca ccg tgc cca gcacct gaa ctc ctg ggg gga ccg tca 1200 His Thr Cys Pro Pro Cys Pro Ala ProGlu Leu Leu Gly Gly Pro Ser 385 390 395 400 gtc ttc ctc ttc ccc cca aaaccc aag gac acc ctc atg atc tcc cgg 1248 Val Phe Leu Phe Pro Pro Lys ProLys Asp Thr Leu Met Ile Ser Arg 405 410 415 acc cct gag gtc aca tgc gtggtg gtg gac gtg agc cac gaa gac cct 1296 Thr Pro Glu Val Thr Cys Val ValVal Asp Val Ser His Glu Asp Pro 420 425 430 gag gtc aag ttc aac tgg tacgtg gac ggc gtg gag gtg cat aat gcc 1344 Glu Val Lys Phe Asn Trp Tyr ValAsp Gly Val Glu Val His Asn Ala 435 440 445 aag aca aag ccg cgg gag gagcag tac aac agc acg tac cgg gtg gtc 1392 Lys Thr Lys Pro Arg Glu Glu GlnTyr Asn Ser Thr Tyr Arg Val Val 450 455 460 agc gtc ctc acc gtc ctg caccag gac tgg ctg aat ggc aag gag tac 1440 Ser Val Leu Thr Val Leu His GlnAsp Trp Leu Asn Gly Lys Glu Tyr 465 470 475 480 aag tgc aag gtc tcc aacaaa gcc ctc cca gcc ccc atc gag aaa acc 1488 Lys Cys Lys Val Ser Asn LysAla Leu Pro Ala Pro Ile Glu Lys Thr 485 490 495 atc tcc aaa gcc aaa gggcag ccc cga gaa cca cag gtg tac acc ctg 1536 Ile Ser Lys Ala Lys Gly GlnPro Arg Glu Pro Gln Val Tyr Thr Leu 500 505 510 ccc cca tcc cgg gat gagctg acc aag aac cag gtc agc ctg acc tgc 1584 Pro Pro Ser Arg Asp Glu LeuThr Lys Asn Gln Val Ser Leu Thr Cys 515 520 525 ctg gtc aaa ggc ttc tatccc agc gac atc gcc gtg gag tgg gag agc 1632 Leu Val Lys Gly Phe Tyr ProSer Asp Ile Ala Val Glu Trp Glu Ser 530 535 540 aat ggg cag ccg gag aacaac tac aag acc acg cct ccc gtg ctg gac 1680 Asn Gly Gln Pro Glu Asn AsnTyr Lys Thr Thr Pro Pro Val Leu Asp 545 550 555 560 tcc gac ggc tcc ttcttc ctc tac agc aag ctc acc gtg gac aag agc 1728 Ser Asp Gly Ser Phe PheLeu Tyr Ser Lys Leu Thr Val Asp Lys Ser 565 570 575 agg tgg cag cag gggaac gtc ttc tca tgc tcc gtg atg cat gag gct 1776 Arg Trp Gln Gln Gly AsnVal Phe Ser Cys Ser Val Met His Glu Ala 580 585 590 ctg cac aac cac tacacg cag aag agc ctc tcc ctg tct ccg ggt aaa 1824 Leu His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro Gly Lys 595 600 605 tga 1827 10 608 PRTHomo sapiens 10 Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu ValLeu Leu 1 5 10 15 Glu Leu Leu Val Gly Ile Tyr Pro Ser Gly Val Ile GlyLeu Val Pro 20 25 30 His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys ProGln Gly Lys 35 40 45 Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr LysCys His Lys 50 55 60 Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly GlnAsp Thr Asp 65 70 75 80 Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala SerGlu Asn His Leu 85 90 95 Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys GluMet Gly Gln Val 100 105 110 Glu Ile Ser Ser Cys Thr Val Asp Arg Asp ThrVal Cys Gly Cys Arg 115 120 125 Lys Asn Gln Tyr Arg His Tyr Trp Ser GluAsn Leu Phe Gln Cys Phe 130 135 140 Asn Cys Ser Leu Cys Leu Asn Gly ThrVal His Leu Ser Cys Gln Glu 145 150 155 160 Lys Gln Asn Thr Val Cys ThrCys His Ala Gly Phe Phe Leu Arg Glu 165 170 175 Asn Glu Cys Val Ser CysSer Asn Cys Lys Lys Ser Asn Glu Thr Asn 180 185 190 Lys Thr Cys Leu HisAsn Gly Ser Arg Glu Lys Asn Asp Ser Val Cys 195 200 205 Pro Gln Gly LysTyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr 210 215 220 Lys Cys HisLys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly 225 230 235 240 GlnAsp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser 245 250 255Glu Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu 260 265270 Met Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val 275280 285 Cys Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu290 295 300 Phe Gln Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly Thr Val HisLeu 305 310 315 320 Ser Cys Gln Glu Lys Gln Asn Thr Val Cys Thr Cys HisAla Gly Phe 325 330 335 Phe Leu Arg Glu Asn Glu Cys Val Ser Cys Ser AsnCys Lys Lys Ser 340 345 350 Leu Glu Cys Thr Lys Leu Cys Leu Pro Gln IleGlu Asn Val Lys Gly 355 360 365 Thr Glu Asp Ser Gly Thr Thr Ala Glu ProLys Ser Cys Asp Lys Thr 370 375 380 His Thr Cys Pro Pro Cys Pro Ala ProGlu Leu Leu Gly Gly Pro Ser 385 390 395 400 Val Phe Leu Phe Pro Pro LysPro Lys Asp Thr Leu Met Ile Ser Arg 405 410 415 Thr Pro Glu Val Thr CysVal Val Val Asp Val Ser His Glu Asp Pro 420 425 430 Glu Val Lys Phe AsnTrp Tyr Val Asp Gly Val Glu Val His Asn Ala 435 440 445 Lys Thr Lys ProArg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 450 455 460 Ser Val LeuThr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 465 470 475 480 LysCys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 485 490 495Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 500 505510 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 515520 525 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser530 535 540 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val LeuAsp 545 550 555 560 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser 565 570 575 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser ValMet His Glu Ala 580 585 590 Leu His Asn His Tyr Thr Gln Lys Ser Leu SerLeu Ser Pro Gly Lys 595 600 605 11 1980 DNA Homo sapiens CDS (1)..(1977)mgTNFR2-TNFR2-IgG 11 atg gcg ccc gtc gcc gtc tgg gcc gcg ctg gcc gtc ggactg gag ctc 48 Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly LeuGlu Leu 1 5 10 15 tgg gct gcg gcg cac gcc ttg ccc gcc cag gtg gca tttaca ccc tac 96 Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe ThrPro Tyr 20 25 30 gcc ccg gag ccc ggg agc aca tgc cgg ctc aga gaa tac tatgac cag 144 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr AspGln 35 40 45 aca gct cag atg tgc tgc agc aaa tgc tcg ccg ggc caa cat gcaaaa 192 Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys50 55 60 gtc ttc tgt acc aag acc tcg gac acc gtg tgt gac tcc tgt gag gac240 Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp 6570 75 80 agc aca tac acc cag ctc tgg aac tgg gtt ccc gag tgc ttg agc tgt288 Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys 8590 95 ggc tcc cgc tgt agc tct gac cag gtg gaa act caa gcc tgc act cgg336 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg 100105 110 gaa cag aac cgc atc tgc acc tgc agg ccc ggc tgg tac tgc gcg ctg384 Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 115120 125 agc aag cag gag ggg tgc cgg ctg tgc gcg ccg ctg cgc aag tgc cgc432 Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg 130135 140 ccg ggc ttc ggc gtg gcc aga cca gga act gaa aca tca gac gtg gtg480 Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val 145150 155 160 tgc aag ccc tgt gcc ccg ggg acg ttc tcc aac acg act tca tccacg 528 Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr165 170 175 gat att tgc agg ccc cac cag atc tgt aac gtg gtg gcc atc cctggg 576 Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly180 185 190 aat gca agc atg gat gca aac tgc acg tcc ccg gag ccc aac agcaca 624 Asn Ala Ser Met Asp Ala Asn Cys Thr Ser Pro Glu Pro Asn Ser Thr195 200 205 tgc cgg ctc aga gaa tac tat gac cag aca gct cag atg tgc tgcagc 672 Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys Ser210 215 220 aaa tgc tcg ccg ggc caa cat gca aaa gtc ttc tgt acc aag acctcg 720 Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr Ser225 230 235 240 gac acc gtg tgt gac tcc tgt gag gac agc aca tac acc cagctc tgg 768 Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln LeuTrp 245 250 255 aac tgg gtt ccc gag tgc ttg agc tgt ggc tcc cgc tgt agctct gac 816 Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser SerAsp 260 265 270 cag gtg gaa act caa gcc tgc act cgg gaa cag aac cgc atctgc acc 864 Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile CysThr 275 280 285 tgc agg ccc ggc tgg tac tgc gcg ctg agc aag cag gag gggtgc cgg 912 Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly CysArg 290 295 300 ctg tgc gcg ccg ctg cgc aag tgc cgc ccg ggc ttc ggc gtggcc aga 960 Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val AlaArg 305 310 315 320 cca gga act gaa aca tca gac gtg gtg tgc aag ccc tgtgcc ccg ggg 1008 Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys AlaPro Gly 325 330 335 acg ttc tcc aac acg act tca tcc acg gat att tgc aggccc cac cag 1056 Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg ProHis Gln 340 345 350 atc tgt aac gtg gtg gcc atc cct ggg aat gca agc atggat gca gtc 1104 Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met AspAla Val 355 360 365 tgc acg tcc acg tcc ccc acc cgg agt atg gcc cca ggggca gta cac 1152 Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly AlaVal His 370 375 380 tta ccc cag cca gtg tcc aca cga tcc caa cac acg cagcca act cca 1200 Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln ProThr Pro 385 390 395 400 gaa ccc agc act gct cca agc acc tcc ttc ctg ctccca atg ggc ccc 1248 Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu ProMet Gly Pro 405 410 415 agc ccc cca gct gaa ggg agc act ggc gac gca gagccc aaa tct tgt 1296 Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Ala Glu ProLys Ser Cys 420 425 430 gac aaa act cac aca tgc cca ccg tgc cca gca cctgaa ctc ctg ggg 1344 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro GluLeu Leu Gly 435 440 445 gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aaggac acc ctc atg 1392 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys AspThr Leu Met 450 455 460 atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtggac gtg agc cac 1440 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val AspVal Ser His 465 470 475 480 gaa gac cct gag gtc aag ttc aac tgg tac gtggac ggc gtg gag gtg 1488 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val AspGly Val Glu Val 485 490 495 cat aat gcc aag aca aag ccg cgg gag gag cagtac aac agc acg tac 1536 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln TyrAsn Ser Thr Tyr 500 505 510 cgg gtg gtc agc gtc ctc acc gtc ctg cac caggac tgg ctg aat ggc 1584 Arg Val Val Ser Val Leu Thr Val Leu His Gln AspTrp Leu Asn Gly 515 520 525 aag gag tac aag tgc aag gtc tcc aac aaa gccctc cca gcc ccc atc 1632 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala LeuPro Ala Pro Ile 530 535 540 gag aaa acc atc tcc aaa gcc aaa ggg cag ccccga gaa cca cag gtg 1680 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro ArgGlu Pro Gln Val 545 550 555 560 tac acc ctg ccc cca tcc cgg gat gag ctgacc aag aac cag gtc agc 1728 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu ThrLys Asn Gln Val Ser 565 570 575 ctg acc tgc ctg gtc aaa ggc ttc tat cccagc gac atc gcc gtg gag 1776 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro SerAsp Ile Ala Val Glu 580 585 590 tgg gag agc aat ggg cag ccg gag aac aactac aag acc acg cct ccc 1824 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn TyrLys Thr Thr Pro Pro 595 600 605 gtg ctg gac tcc gac ggc tcc ttc ttc ctctac agc aag ctc acc gtg 1872 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu TyrSer Lys Leu Thr Val 610 615 620 gac aag agc agg tgg cag cag ggg aac gtcttc tca tgc tcc gtg atg 1920 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val PheSer Cys Ser Val Met 625 630 635 640 cat gag gct ctg cac aac cac tac acgcag aag agc ctc tcc ctg tct 1968 His Glu Ala Leu His Asn His Tyr Thr GlnLys Ser Leu Ser Leu Ser 645 650 655 ccg ggt aaa tga 1980 Pro Gly Lys 12659 PRT Homo sapiens 12 Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala ValGly Leu Glu Leu 1 5 10 15 Trp Ala Ala Ala His Ala Leu Pro Ala Gln ValAla Phe Thr Pro Tyr 20 25 30 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu ArgGlu Tyr Tyr Asp Gln 35 40 45 Thr Ala Gln Met Cys Cys Ser Lys Cys Ser ProGly Gln His Ala Lys 50 55 60 Val Phe Cys Thr Lys Thr Ser Asp Thr Val CysAsp Ser Cys Glu Asp 65 70 75 80 Ser Thr Tyr Thr Gln Leu Trp Asn Trp ValPro Glu Cys Leu Ser Cys 85 90 95 Gly Ser Arg Cys Ser Ser Asp Gln Val GluThr Gln Ala Cys Thr Arg 100 105 110 Glu Gln Asn Arg Ile Cys Thr Cys ArgPro Gly Trp Tyr Cys Ala Leu 115 120 125 Ser Lys Gln Glu Gly Cys Arg LeuCys Ala Pro Leu Arg Lys Cys Arg 130 135 140 Pro Gly Phe Gly Val Ala ArgPro Gly Thr Glu Thr Ser Asp Val Val 145 150 155 160 Cys Lys Pro Cys AlaPro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr 165 170 175 Asp Ile Cys ArgPro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly 180 185 190 Asn Ala SerMet Asp Ala Asn Cys Thr Ser Pro Glu Pro Asn Ser Thr 195 200 205 Cys ArgLeu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys Ser 210 215 220 LysCys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr Ser 225 230 235240 Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp 245250 255 Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp260 265 270 Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile CysThr 275 280 285 Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu GlyCys Arg 290 295 300 Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe GlyVal Ala Arg 305 310 315 320 Pro Gly Thr Glu Thr Ser Asp Val Val Cys LysPro Cys Ala Pro Gly 325 330 335 Thr Phe Ser Asn Thr Thr Ser Ser Thr AspIle Cys Arg Pro His Gln 340 345 350 Ile Cys Asn Val Val Ala Ile Pro GlyAsn Ala Ser Met Asp Ala Val 355 360 365 Cys Thr Ser Thr Ser Pro Thr ArgSer Met Ala Pro Gly Ala Val His 370 375 380 Leu Pro Gln Pro Val Ser ThrArg Ser Gln His Thr Gln Pro Thr Pro 385 390 395 400 Glu Pro Ser Thr AlaPro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro 405 410 415 Ser Pro Pro AlaGlu Gly Ser Thr Gly Asp Ala Glu Pro Lys Ser Cys 420 425 430 Asp Lys ThrHis Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 435 440 445 Gly ProSer Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 450 455 460 IleSer Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 465 470 475480 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 485490 495 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr500 505 510 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly 515 520 525 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro AlaPro Ile 530 535 540 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg GluPro Gln Val 545 550 555 560 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu ThrLys Asn Gln Val Ser 565 570 575 Leu Thr Cys Leu Val Lys Gly Phe Tyr ProSer Asp Ile Ala Val Glu 580 585 590 Trp Glu Ser Asn Gly Gln Pro Glu AsnAsn Tyr Lys Thr Thr Pro Pro 595 600 605 Val Leu Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val 610 615 620 Asp Lys Ser Arg Trp Gln GlnGly Asn Val Phe Ser Cys Ser Val Met 625 630 635 640 His Glu Ala Leu HisAsn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 645 650 655 Pro Gly Lys 131314 DNA Homo sapiens CDS (1)..(1311) CD2-IgG 13 atg agc ttt cca tgt aaattt gta gcc agc ttc ctt ctg att ttc aat 48 Met Ser Phe Pro Cys Lys PheVal Ala Ser Phe Leu Leu Ile Phe Asn 1 5 10 15 gtt tct tcc aaa ggt gcagtc tcc aaa gag att acg aat gcc ttg gaa 96 Val Ser Ser Lys Gly Ala ValSer Lys Glu Ile Thr Asn Ala Leu Glu 20 25 30 acc tgg ggt gcc ttg ggt caggac atc aac ttg gac att cct agt ttt 144 Thr Trp Gly Ala Leu Gly Gln AspIle Asn Leu Asp Ile Pro Ser Phe 35 40 45 caa atg agt gat gat att gac gatata aaa tgg gaa aaa act tca gac 192 Gln Met Ser Asp Asp Ile Asp Asp IleLys Trp Glu Lys Thr Ser Asp 50 55 60 aag aaa aag att gca caa ttc aga aaagag aaa gag act ttc aag gaa 240 Lys Lys Lys Ile Ala Gln Phe Arg Lys GluLys Glu Thr Phe Lys Glu 65 70 75 80 aaa gat aca tat aag cta ttt aaa aatgga act ctg aaa att aag cat 288 Lys Asp Thr Tyr Lys Leu Phe Lys Asn GlyThr Leu Lys Ile Lys His 85 90 95 ctg aag acc gat gat cag gat atc tac aaggta tca ata tat gat aca 336 Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys ValSer Ile Tyr Asp Thr 100 105 110 aaa gga aaa aat gtg ttg gaa aaa ata tttgat ttg aag att caa gag 384 Lys Gly Lys Asn Val Leu Glu Lys Ile Phe AspLeu Lys Ile Gln Glu 115 120 125 agg gtc tca aaa cca aag atc tcc tgg acttgt atc aac aca acc ctg 432 Arg Val Ser Lys Pro Lys Ile Ser Trp Thr CysIle Asn Thr Thr Leu 130 135 140 acc tgt gag gta atg aat gga act gac cccgaa tta aac ctg tat caa 480 Thr Cys Glu Val Met Asn Gly Thr Asp Pro GluLeu Asn Leu Tyr Gln 145 150 155 160 gat ggg aaa cat cta aaa ctt tct cagagg gtc atc aca cac aag tgg 528 Asp Gly Lys His Leu Lys Leu Ser Gln ArgVal Ile Thr His Lys Trp 165 170 175 acc acc agc ctg agt gca aaa ttc aagtgc aca gca ggg aac aaa gtc 576 Thr Thr Ser Leu Ser Ala Lys Phe Lys CysThr Ala Gly Asn Lys Val 180 185 190 agc aag gaa tcc agt gtc gag cct gtcagc tgt cct gca gag ccc aaa 624 Ser Lys Glu Ser Ser Val Glu Pro Val SerCys Pro Ala Glu Pro Lys 195 200 205 tct tgt gac aaa act cac aca tgc ccaccg tgc cca gca cct gaa ctc 672 Ser Cys Asp Lys Thr His Thr Cys Pro ProCys Pro Ala Pro Glu Leu 210 215 220 ctg ggg gga ccg tca gtc ttc ctc ttcccc cca aaa ccc aag gac acc 720 Leu Gly Gly Pro Ser Val Phe Leu Phe ProPro Lys Pro Lys Asp Thr 225 230 235 240 ctc atg atc tcc cgg acc cct gaggtc aca tgc gtg gtg gtg gac gtg 768 Leu Met Ile Ser Arg Thr Pro Glu ValThr Cys Val Val Val Asp Val 245 250 255 agc cac gaa gac cct gag gtc aagttc aac tgg tac gtg gac ggc gtg 816 Ser His Glu Asp Pro Glu Val Lys PheAsn Trp Tyr Val Asp Gly Val 260 265 270 gag gtg cat aat gcc aag aca aagccg cgg gag gag cag tac aac agc 864 Glu Val His Asn Ala Lys Thr Lys ProArg Glu Glu Gln Tyr Asn Ser 275 280 285 acg tac cgg gtg gtc agc gtc ctcacc gtc ctg cac cag gac tgg ctg 912 Thr Tyr Arg Val Val Ser Val Leu ThrVal Leu His Gln Asp Trp Leu 290 295 300 aat ggc aag gag tac aag tgc aaggtc tcc aac aaa gcc ctc cca gcc 960 Asn Gly Lys Glu Tyr Lys Cys Lys ValSer Asn Lys Ala Leu Pro Ala 305 310 315 320 ccc atc gag aaa acc atc tccaaa gcc aaa ggg cag ccc cga gaa cca 1008 Pro Ile Glu Lys Thr Ile Ser LysAla Lys Gly Gln Pro Arg Glu Pro 325 330 335 cag gtg tac acc ctg ccc ccatcc cgg gat gag ctg acc aag aac cag 1056 Gln Val Tyr Thr Leu Pro Pro SerArg Asp Glu Leu Thr Lys Asn Gln 340 345 350 gtc agc ctg acc tgc ctg gtcaaa ggc ttc tat ccc agc gac atc gcc 1104 Val Ser Leu Thr Cys Leu Val LysGly Phe Tyr Pro Ser Asp Ile Ala 355 360 365 gtg gag tgg gag agc aat gggcag ccg gag aac aac tac aag acc acg 1152 Val Glu Trp Glu Ser Asn Gly GlnPro Glu Asn Asn Tyr Lys Thr Thr 370 375 380 cct ccc gtg ctg gac tcc gacggc tcc ttc ttc ctc tac agc aag ctc 1200 Pro Pro Val Leu Asp Ser Asp GlySer Phe Phe Leu Tyr Ser Lys Leu 385 390 395 400 acc gtg gac aag agc aggtgg cag cag ggg aac gtc ttc tca tgc tcc 1248 Thr Val Asp Lys Ser Arg TrpGln Gln Gly Asn Val Phe Ser Cys Ser 405 410 415 gtg atg cat gag gct ctgcac aac cac tac acg cag aag agc ctc tcc 1296 Val Met His Glu Ala Leu HisAsn His Tyr Thr Gln Lys Ser Leu Ser 420 425 430 ctg tct ccg ggt aaa tga1314 Leu Ser Pro Gly Lys 435 14 437 PRT Homo sapiens 14 Met Ser Phe ProCys Lys Phe Val Ala Ser Phe Leu Leu Ile Phe Asn 1 5 10 15 Val Ser SerLys Gly Ala Val Ser Lys Glu Ile Thr Asn Ala Leu Glu 20 25 30 Thr Trp GlyAla Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro Ser Phe 35 40 45 Gln Met SerAsp Asp Ile Asp Asp Ile Lys Trp Glu Lys Thr Ser Asp 50 55 60 Lys Lys LysIle Ala Gln Phe Arg Lys Glu Lys Glu Thr Phe Lys Glu 65 70 75 80 Lys AspThr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile Lys His 85 90 95 Leu LysThr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp Thr 100 105 110 LysGly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys Ile Gln Glu 115 120 125Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile Asn Thr Thr Leu 130 135140 Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu Asn Leu Tyr Gln 145150 155 160 Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val Ile Thr His LysTrp 165 170 175 Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys Thr Ala Gly AsnLys Val 180 185 190 Ser Lys Glu Ser Ser Val Glu Pro Val Ser Cys Pro AlaGlu Pro Lys 195 200 205 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys ProAla Pro Glu Leu 210 215 220 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro ProLys Pro Lys Asp Thr 225 230 235 240 Leu Met Ile Ser Arg Thr Pro Glu ValThr Cys Val Val Val Asp Val 245 250 255 Ser His Glu Asp Pro Glu Val LysPhe Asn Trp Tyr Val Asp Gly Val 260 265 270 Glu Val His Asn Ala Lys ThrLys Pro Arg Glu Glu Gln Tyr Asn Ser 275 280 285 Thr Tyr Arg Val Val SerVal Leu Thr Val Leu His Gln Asp Trp Leu 290 295 300 Asn Gly Lys Glu TyrLys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 305 310 315 320 Pro Ile GluLys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 325 330 335 Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 340 345 350 ValSer Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 355 360 365Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 370 375380 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 385390 395 400 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser CysSer 405 410 415 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys SerLeu Ser 420 425 430 Leu Ser Pro Gly Lys 435 15 1134 DNA Homo sapiens CDS(1)..(1131) CTLA4-IgG 15 atg agg acc tgg ccc tgc act ctc ctg ttt ttt cttctc ttc atc cct 48 Met Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu LeuPhe Ile Pro 1 5 10 15 gtc ttc tgc aaa gca atg cac gtg gcc cag cct gctgtg gta ctg gcc 96 Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala ValVal Leu Ala 20 25 30 agc agc cga ggc atc gcc agc ttt gtg tgt gag tat gcatct cca ggc 144 Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala SerPro Gly 35 40 45 aaa gcc act gag gtc cgg gtg aca gtg ctt cgg cag gct gacagc cag 192 Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp SerGln 50 55 60 gtg act gaa gtc tgt gcg gca acc tac atg atg ggg aat gag ttgacc 240 Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr65 70 75 80 ttc cta gat gat tcc atc tgc acg ggc acc tcc agt gga aat caagtg 288 Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val85 90 95 aac ctc act atc caa gga ctg agg gcc atg gac acg gga ctc tac atc336 Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile 100105 110 tgc aag gtg gag ctc atg tac cca ccg cca tac tac ctg ggc ata ggc384 Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly 115120 125 aac gga acc cag att tat gta att gat cca gaa ccg tgc cca gat tct432 Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser 130135 140 gca gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca480 Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 145150 155 160 gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc ccaaaa 528 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys165 170 175 ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgcgtg 576 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val180 185 190 gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tggtac 624 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr195 200 205 gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gaggag 672 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu210 215 220 cag tac aac agc acg tac cgg gtg gtc agc gtc ctc acc gtc ctgcac 720 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His225 230 235 240 cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tccaac aaa 768 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser AsnLys 245 250 255 gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaaggg cag 816 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln 260 265 270 ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gatgag ctg 864 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp GluLeu 275 280 285 acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttctat ccc 912 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe TyrPro 290 295 300 agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gagaac aac 960 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu AsnAsn 305 310 315 320 tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tccttc ttc ctc 1008 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser PhePhe Leu 325 330 335 tac agc aag ctc acc gtg gac aag agc agg tgg cag cagggg aac gtc 1056 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln GlyAsn Val 340 345 350 ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cactac acg cag 1104 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His TyrThr Gln 355 360 365 aag agc ctc tcc ctg tct ccg ggt aaa tga 1134 Lys SerLeu Ser Leu Ser Pro Gly Lys 370 375 16 377 PRT Homo sapiens 16 Met ArgThr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro 1 5 10 15 ValPhe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala 20 25 30 SerSer Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly 35 40 45 LysAla Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln 50 55 60 ValThr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr 65 70 75 80Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val 85 90 95Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile 100 105110 Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly 115120 125 Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser130 135 140 Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro CysPro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu PhePro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro GluVal Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu ValLys Phe Asn Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala LysThr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Tyr Arg Val ValSer Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly LysGlu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln 260 265 270 Pro Arg Glu Pro GlnVal Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 275 280 285 Thr Lys Asn GlnVal Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp IleAla Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 305 310 315 320 TyrLys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 340 345350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 355360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 17 1854 DNA Homosapiens CDS (1)..(1851) CD2-CD2-IgG 17 atg agc ttt cca tgt aaa ttt gtagcc agc ttc ctt ctg att ttc aat 48 Met Ser Phe Pro Cys Lys Phe Val AlaSer Phe Leu Leu Ile Phe Asn 1 5 10 15 gtt tct tcc aaa ggt gca gtc tccaaa gag att acg aat gcc ttg gaa 96 Val Ser Ser Lys Gly Ala Val Ser LysGlu Ile Thr Asn Ala Leu Glu 20 25 30 acc tgg ggt gcc ttg ggt cag gac atcaac ttg gac att cct agt ttt 144 Thr Trp Gly Ala Leu Gly Gln Asp Ile AsnLeu Asp Ile Pro Ser Phe 35 40 45 caa atg agt gat gat att gac gat ata aaatgg gaa aaa act tca gac 192 Gln Met Ser Asp Asp Ile Asp Asp Ile Lys TrpGlu Lys Thr Ser Asp 50 55 60 aag aaa aag att gca caa ttc aga aaa gag aaagag act ttc aag gaa 240 Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys GluThr Phe Lys Glu 65 70 75 80 aaa gat aca tat aag cta ttt aaa aat gga actctg aaa att aag cat 288 Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr LeuLys Ile Lys His 85 90 95 ctg aag acc gat gat cag gat atc tac aag gta tcaata tat gat aca 336 Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser IleTyr Asp Thr 100 105 110 aaa gga aaa aat gtg ttg gaa aaa ata ttt gat ttgaag att caa gag 384 Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu LysIle Gln Glu 115 120 125 agg gtc tca aaa cca aag atc tcc tgg act tgt atcaac aca acc ctg 432 Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile AsnThr Thr Leu 130 135 140 acc tgt gag gta atg aat gga act gac ccc gaa ttaaac ctg tat caa 480 Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu AsnLeu Tyr Gln 145 150 155 160 gat ggg aaa cat cta aaa ctt tct cag agg gtcatc aca cac aag tgg 528 Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val IleThr His Lys Trp 165 170 175 acc acc agc ctg agt gca aaa ttc aag tgc acagca ggg aac aaa gtc 576 Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys Thr AlaGly Asn Lys Val 180 185 190 agc aag gaa tcc agt gtc gag cct gtc agc tgtcct aaa gag att acg 624 Ser Lys Glu Ser Ser Val Glu Pro Val Ser Cys ProLys Glu Ile Thr 195 200 205 aat gcc ttg gaa acc tgg ggt gcc ttg ggt caggac atc aac ttg gac 672 Asn Ala Leu Glu Thr Trp Gly Ala Leu Gly Gln AspIle Asn Leu Asp 210 215 220 att cct agt ttt caa atg agt gat gat att gacgat ata aaa tgg gaa 720 Ile Pro Ser Phe Gln Met Ser Asp Asp Ile Asp AspIle Lys Trp Glu 225 230 235 240 aaa act tca gac aag aaa aag att gca caattc aga aaa gag aaa gag 768 Lys Thr Ser Asp Lys Lys Lys Ile Ala Gln PheArg Lys Glu Lys Glu 245 250 255 act ttc aag gaa aaa gat aca tat aag ctattt aaa aat gga act ctg 816 Thr Phe Lys Glu Lys Asp Thr Tyr Lys Leu PheLys Asn Gly Thr Leu 260 265 270 aaa att aag cat ctg aag acc gat gat caggat atc tac aag gta tca 864 Lys Ile Lys His Leu Lys Thr Asp Asp Gln AspIle Tyr Lys Val Ser 275 280 285 ata tat gat aca aaa gga aaa aat gtg ttggaa aaa ata ttt gat ttg 912 Ile Tyr Asp Thr Lys Gly Lys Asn Val Leu GluLys Ile Phe Asp Leu 290 295 300 aag att caa gag agg gtc tca aaa cca aagatc tcc tgg act tgt atc 960 Lys Ile Gln Glu Arg Val Ser Lys Pro Lys IleSer Trp Thr Cys Ile 305 310 315 320 aac aca acc ctg acc tgt gag gta atgaat gga act gac ccc gaa tta 1008 Asn Thr Thr Leu Thr Cys Glu Val Met AsnGly Thr Asp Pro Glu Leu 325 330 335 aac ctg tat caa gat ggg aaa cat ctaaaa ctt tct cag agg gtc atc 1056 Asn Leu Tyr Gln Asp Gly Lys His Leu LysLeu Ser Gln Arg Val Ile 340 345 350 aca cac aag tgg acc acc agc ctg agtgca aaa ttc aag tgc aca gca 1104 Thr His Lys Trp Thr Thr Ser Leu Ser AlaLys Phe Lys Cys Thr Ala 355 360 365 ggg aac aaa gtc agc aag gaa tcc agtgtc gag cct gtc agc tgt cct 1152 Gly Asn Lys Val Ser Lys Glu Ser Ser ValGlu Pro Val Ser Cys Pro 370 375 380 gca gag ccc aaa tct tgt gac aaa actcac aca tgc cca ccg tgc cca 1200 Ala Glu Pro Lys Ser Cys Asp Lys Thr HisThr Cys Pro Pro Cys Pro 385 390 395 400 gca cct gaa ctc ctg ggg gga ccgtca gtc ttc ctc ttc ccc cca aaa 1248 Ala Pro Glu Leu Leu Gly Gly Pro SerVal Phe Leu Phe Pro Pro Lys 405 410 415 ccc aag gac acc ctc atg atc tcccgg acc cct gag gtc aca tgc gtg 1296 Pro Lys Asp Thr Leu Met Ile Ser ArgThr Pro Glu Val Thr Cys Val 420 425 430 gtg gtg gac gtg agc cac gaa gaccct gag gtc aag ttc aac tgg tac 1344 Val Val Asp Val Ser His Glu Asp ProGlu Val Lys Phe Asn Trp Tyr 435 440 445 gtg gac ggc gtg gag gtg cat aatgcc aag aca aag ccg cgg gag gag 1392 Val Asp Gly Val Glu Val His Asn AlaLys Thr Lys Pro Arg Glu Glu 450 455 460 cag tac aac agc acg tac cgg gtggtc agc gtc ctc acc gtc tgt cac 1440 Gln Tyr Asn Ser Thr Tyr Arg Val ValSer Val Leu Thr Val Cys His 465 470 475 480 cag gac tgg ctg aat ggc aaggag tac aag tgc aag gtc tcc aac aaa 1488 Gln Asp Trp Leu Asn Gly Lys GluTyr Lys Cys Lys Val Ser Asn Lys 485 490 495 gcc ctc cca gcc ccc atc gagaaa acc atc tcc aaa gcc aaa ggg cag 1536 Ala Leu Pro Ala Pro Ile Glu LysThr Ile Ser Lys Ala Lys Gly Gln 500 505 510 ccc cga gaa cca cag gtg tacacc ctg ccc cca tcc cgg gat gag ctg 1584 Pro Arg Glu Pro Gln Val Tyr ThrLeu Pro Pro Ser Arg Asp Glu Leu 515 520 525 acc aag aac cag gtc agc ctgacc tgc ctg gtc aaa ggc ttc tat ccc 1632 Thr Lys Asn Gln Val Ser Leu ThrCys Leu Val Lys Gly Phe Tyr Pro 530 535 540 agc gac atc gcc gtg gag tgggag agc aat ggg cag ccg gag aac aac 1680 Ser Asp Ile Ala Val Glu Trp GluSer Asn Gly Gln Pro Glu Asn Asn 545 550 555 560 tac aag acc acg cct cccgtg ctg gac tcc gac ggc tcc ttc ttc ctc 1728 Tyr Lys Thr Thr Pro Pro ValLeu Asp Ser Asp Gly Ser Phe Phe Leu 565 570 575 tac agc aag ctc acc gtggac aag agc agg tgg cag cag ggg aac gtc 1776 Tyr Ser Lys Leu Thr Val AspLys Ser Arg Trp Gln Gln Gly Asn Val 580 585 590 ttc tca tgc tcc gtg atgcat gag gct ctg cac aac cac tac acg cag 1824 Phe Ser Cys Ser Val Met HisGlu Ala Leu His Asn His Tyr Thr Gln 595 600 605 aag agc ctc tcc ctg tctccg ggt aaa tga 1854 Lys Ser Leu Ser Leu Ser Pro Gly Lys 610 615 18 617PRT Homo sapiens 18 Met Ser Phe Pro Cys Lys Phe Val Ala Ser Phe Leu LeuIle Phe Asn 1 5 10 15 Val Ser Ser Lys Gly Ala Val Ser Lys Glu Ile ThrAsn Ala Leu Glu 20 25 30 Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu AspIle Pro Ser Phe 35 40 45 Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp GluLys Thr Ser Asp 50 55 60 Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys GluThr Phe Lys Glu 65 70 75 80 Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly ThrLeu Lys Ile Lys His 85 90 95 Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys ValSer Ile Tyr Asp Thr 100 105 110 Lys Gly Lys Asn Val Leu Glu Lys Ile PheAsp Leu Lys Ile Gln Glu 115 120 125 Arg Val Ser Lys Pro Lys Ile Ser TrpThr Cys Ile Asn Thr Thr Leu 130 135 140 Thr Cys Glu Val Met Asn Gly ThrAsp Pro Glu Leu Asn Leu Tyr Gln 145 150 155 160 Asp Gly Lys His Leu LysLeu Ser Gln Arg Val Ile Thr His Lys Trp 165 170 175 Thr Thr Ser Leu SerAla Lys Phe Lys Cys Thr Ala Gly Asn Lys Val 180 185 190 Ser Lys Glu SerSer Val Glu Pro Val Ser Cys Pro Lys Glu Ile Thr 195 200 205 Asn Ala LeuGlu Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp 210 215 220 Ile ProSer Phe Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu 225 230 235 240Lys Thr Ser Asp Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu 245 250255 Thr Phe Lys Glu Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu 260265 270 Lys Ile Lys His Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser275 280 285 Ile Tyr Asp Thr Lys Gly Lys Asn Val Leu Glu Lys Ile Phe AspLeu 290 295 300 Lys Ile Gln Glu Arg Val Ser Lys Pro Lys Ile Ser Trp ThrCys Ile 305 310 315 320 Asn Thr Thr Leu Thr Cys Glu Val Met Asn Gly ThrAsp Pro Glu Leu 325 330 335 Asn Leu Tyr Gln Asp Gly Lys His Leu Lys LeuSer Gln Arg Val Ile 340 345 350 Thr His Lys Trp Thr Thr Ser Leu Ser AlaLys Phe Lys Cys Thr Ala 355 360 365 Gly Asn Lys Val Ser Lys Glu Ser SerVal Glu Pro Val Ser Cys Pro 370 375 380 Ala Glu Pro Lys Ser Cys Asp LysThr His Thr Cys Pro Pro Cys Pro 385 390 395 400 Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys 405 410 415 Pro Lys Asp Thr LeuMet Ile Ser Arg Thr Pro Glu Val Thr Cys Val 420 425 430 Val Val Asp ValSer His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 435 440 445 Val Asp GlyVal Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 450 455 460 Gln TyrAsn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Cys His 465 470 475 480Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 485 490495 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 500505 510 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu515 520 525 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe TyrPro 530 535 540 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro GluAsn Asn 545 550 555 560 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp GlySer Phe Phe Leu 565 570 575 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg TrpGln Gln Gly Asn Val 580 585 590 Phe Ser Cys Ser Val Met His Glu Ala LeuHis Asn His Tyr Thr Gln 595 600 605 Lys Ser Leu Ser Leu Ser Pro Gly Lys610 615 19 1509 DNA Homo sapiens CDS (1)..(1506) CTLA4-CTLA4-IgG 19 atgagg acc tgg ccc tgc act ctc ctg ttt ttt ctt ctc ttc atc cct 48 Met ArgThr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro 1 5 10 15 gtcttc tgc aaa gca atg cac gtg gcc cag cct gct gtg gta ctg gcc 96 Val PheCys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala 20 25 30 agc agccga ggc atc gcc agc ttt gtg tgt gag tat gca tct cca ggc 144 Ser Ser ArgGly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly 35 40 45 aaa gcc actgag gtc cgg gtg aca gtg ctt cgg cag gct gac agc cag 192 Lys Ala Thr GluVal Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln 50 55 60 gtg act gaa gtctgt gcg gca acc tac atg atg ggg aat gag ttg acc 240 Val Thr Glu Val CysAla Ala Thr Tyr Met Met Gly Asn Glu Leu Thr 65 70 75 80 ttc cta gat gattcc atc tgc acg ggc acc tcc agt gga aat caa gtg 288 Phe Leu Asp Asp SerIle Cys Thr Gly Thr Ser Ser Gly Asn Gln Val 85 90 95 aac ctc act atc caagga ctg agg gcc atg gac acg gga ctc tac atc 336 Asn Leu Thr Ile Gln GlyLeu Arg Ala Met Asp Thr Gly Leu Tyr Ile 100 105 110 tgc aag gtg gag ctcatg tac cca ccg cca tac tac ctg ggc ata ggc 384 Cys Lys Val Glu Leu MetTyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly 115 120 125 aac gga acc cag atttat gta att gat cca gaa ccg tgc cca gat tcg 432 Asn Gly Thr Gln Ile TyrVal Ile Asp Pro Glu Pro Cys Pro Asp Ser 130 135 140 gat aac atg cac gtggcc cag cct gct gtg gta ctg gcc agc agc cga 480 Asp Asn Met His Val AlaGln Pro Ala Val Val Leu Ala Ser Ser Arg 145 150 155 160 ggc atc gcc agcttt gtg tgt gag tat gca tct cca ggc aaa gcc act 528 Gly Ile Ala Ser PheVal Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr 165 170 175 gag gtc cgg gtgaca gtg ctt cgg cag gct gac agc cag gtg act gaa 576 Glu Val Arg Val ThrVal Leu Arg Gln Ala Asp Ser Gln Val Thr Glu 180 185 190 gtc tgt gcg gcaacc tac atg atg ggg aat gag ttg acc ttc cta gat 624 Val Cys Ala Ala ThrTyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp 195 200 205 gat tcc atc tgcacg ggc acc tcc agt gga aat caa gtg aac ctc act 672 Asp Ser Ile Cys ThrGly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr 210 215 220 atc caa gga ctgagg gcc atg gac acg gga ctc tac atc tgc aag gtg 720 Ile Gln Gly Leu ArgAla Met Asp Thr Gly Leu Tyr Ile Cys Lys Val 225 230 235 240 gag ctc atgtac cca ccg cca tac tac ctg ggc ata ggc aac gga acc 768 Glu Leu Met TyrPro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr 245 250 255 cag att tatgta att gat cca gaa ccg tgc cca gat tct gca gag ccc 816 Gln Ile Tyr ValIle Asp Pro Glu Pro Cys Pro Asp Ser Ala Glu Pro 260 265 270 aaa tct tgtgac aaa act cac aca tgc cca ccg tgc cca gca cct gaa 864 Lys Ser Cys AspLys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 275 280 285 ctc ctg ggggga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 912 Leu Leu Gly GlyPro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 290 295 300 acc ctc atgatc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 960 Thr Leu Met IleSer Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 305 310 315 320 gtg agccac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 1008 Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 325 330 335 gtg gaggtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac 1056 Val Glu ValHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 340 345 350 agc acgtac cgg gtg gtc agc gtc ctc acc gtc tgt cac cag gac tgg 1104 Ser Thr TyrArg Val Val Ser Val Leu Thr Val Cys His Gln Asp Trp 355 360 365 ctg aatggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1152 Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 370 375 380 gcc cccatc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa 1200 Ala Pro IleGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 385 390 395 400 ccacag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac 1248 Pro GlnVal Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 405 410 415 caggtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1296 Gln ValSer Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 420 425 430 gccgtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc 1344 Ala ValGlu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 435 440 445 acgcct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1392 Thr ProPro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 450 455 460 ctcacc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc 1440 Leu ThrVal Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 465 470 475 480tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc 1488 SerVal Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 485 490 495tcc ctg tct ccg ggt aaa tga 1509 Ser Leu Ser Pro Gly Lys 500 20 502 PRTHomo sapiens 20 Met Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu PheIle Pro 1 5 10 15 Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala ValVal Leu Ala 20 25 30 Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr AlaSer Pro Gly 35 40 45 Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln AlaAsp Ser Gln 50 55 60 Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly AsnGlu Leu Thr 65 70 75 80 Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser SerGly Asn Gln Val 85 90 95 Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp ThrGly Leu Tyr Ile 100 105 110 Cys Lys Val Glu Leu Met Tyr Pro Pro Pro TyrTyr Leu Gly Ile Gly 115 120 125 Asn Gly Thr Gln Ile Tyr Val Ile Asp ProGlu Pro Cys Pro Asp Ser 130 135 140 Asp Asn Met His Val Ala Gln Pro AlaVal Val Leu Ala Ser Ser Arg 145 150 155 160 Gly Ile Ala Ser Phe Val CysGlu Tyr Ala Ser Pro Gly Lys Ala Thr 165 170 175 Glu Val Arg Val Thr ValLeu Arg Gln Ala Asp Ser Gln Val Thr Glu 180 185 190 Val Cys Ala Ala ThrTyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp 195 200 205 Asp Ser Ile CysThr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr 210 215 220 Ile Gln GlyLeu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val 225 230 235 240 GluLeu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr 245 250 255Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Ala Glu Pro 260 265270 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 275280 285 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp290 295 300 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val ValAsp 305 310 315 320 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp TyrVal Asp Gly 325 330 335 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg GluGlu Gln Tyr Asn 340 345 350 Ser Thr Tyr Arg Val Val Ser Val Leu Thr ValCys His Gln Asp Trp 355 360 365 Leu Asn Gly Lys Glu Tyr Lys Cys Lys ValSer Asn Lys Ala Leu Pro 370 375 380 Ala Pro Ile Glu Lys Thr Ile Ser LysAla Lys Gly Gln Pro Arg Glu 385 390 395 400 Pro Gln Val Tyr Thr Leu ProPro Ser Arg Asp Glu Leu Thr Lys Asn 405 410 415 Gln Val Ser Leu Thr CysLeu Val Lys Gly Phe Tyr Pro Ser Asp Ile 420 425 430 Ala Val Glu Trp GluSer Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 435 440 445 Thr Pro Pro ValLeu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 450 455 460 Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 465 470 475 480 SerVal Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 485 490 495Ser Leu Ser Pro Gly Lys 500 21 1854 DNA Homo sapiens CDS (1)..(1851)mgCD2-CD2-IgG 21 atg agc ttt cca tgt aaa ttt gta gcc agc ttc ctt ctg attttc aat 48 Met Ser Phe Pro Cys Lys Phe Val Ala Ser Phe Leu Leu Ile PheAsn 1 5 10 15 gtt tct tcc aaa ggt gca gtc tcc aaa gag att acg aat gccttg gaa 96 Val Ser Ser Lys Gly Ala Val Ser Lys Glu Ile Thr Asn Ala LeuGlu 20 25 30 acc tgg ggt gcc ttg ggt cag gac atc aac ttg gac att cct agtttt 144 Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro Ser Phe35 40 45 caa atg agt gat gat att gac gat ata aaa tgg gaa aaa act tca gac192 Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys Thr Ser Asp 5055 60 aag aaa aag att gca caa ttc aga aaa gag aaa gag act ttc aag gaa240 Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu Thr Phe Lys Glu 6570 75 80 aaa gat aca tat aag cta ttt aaa aat gga act ctg aaa att aag cat288 Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile Lys His 8590 95 ctg aag acc gat gat cag gat atc tac aag gta tca ata tat gat aca336 Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp Thr 100105 110 aaa gga aaa aat gtg ttg gaa aaa ata ttt gat ttg aag att caa gag384 Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys Ile Gln Glu 115120 125 agg gtc tca aaa cca aag atc tcc tgg act tgt atc aac aca acc ctg432 Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile Asn Thr Thr Leu 130135 140 acc tgt gag gta atg aat gga act gac ccc gaa tta aac ctg tat caa480 Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu Asn Leu Tyr Gln 145150 155 160 gat ggg aaa cat cta aaa ctt tct cag agg gtc atc aca cac aagtgg 528 Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val Ile Thr His Lys Trp165 170 175 acc acc agc ctg agt gca aaa ttc aag tgc aca gca ggg aac aaagtc 576 Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys Thr Ala Gly Asn Lys Val180 185 190 agc aag gaa tcc agt gtc gag aat gtc agc tgt cct aaa aat attacg 624 Ser Lys Glu Ser Ser Val Glu Asn Val Ser Cys Pro Lys Asn Ile Thr195 200 205 aat gcc ttg gaa acc tgg ggt gcc ttg ggt cag gac atc aac ttggac 672 Asn Ala Leu Glu Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp210 215 220 att cct agt ttt caa atg agt gat gat att gac gat ata aaa tgggaa 720 Ile Pro Ser Phe Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu225 230 235 240 aaa act tca gac aag aaa aag att gca caa ttc aga aaa gagaaa gag 768 Lys Thr Ser Asp Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu LysGlu 245 250 255 act ttc aag gaa aaa gat aca tat aag cta ttt aaa aat ggaact ctg 816 Thr Phe Lys Glu Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly ThrLeu 260 265 270 aaa att aag cat ctg aag acc gat gat cag gat atc tac aaggta tca 864 Lys Ile Lys His Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys ValSer 275 280 285 ata tat gat aca aaa gga aaa aat gtg ttg gaa aaa ata tttgat ttg 912 Ile Tyr Asp Thr Lys Gly Lys Asn Val Leu Glu Lys Ile Phe AspLeu 290 295 300 aag att caa gag agg gtc tca aaa cca aag atc tcc tgg acttgt atc 960 Lys Ile Gln Glu Arg Val Ser Lys Pro Lys Ile Ser Trp Thr CysIle 305 310 315 320 aac aca acc ctg acc tgt gag gta atg aat gga act gacccc gaa tta 1008 Asn Thr Thr Leu Thr Cys Glu Val Met Asn Gly Thr Asp ProGlu Leu 325 330 335 aac ctg tat caa gat ggg aaa cat cta aaa ctt tct cagagg gtc atc 1056 Asn Leu Tyr Gln Asp Gly Lys His Leu Lys Leu Ser Gln ArgVal Ile 340 345 350 aca cac aag tgg acc acc agc ctg agt gca aaa ttc aagtgc aca gca 1104 Thr His Lys Trp Thr Thr Ser Leu Ser Ala Lys Phe Lys CysThr Ala 355 360 365 ggg aac aaa gtc agc aag gaa tcc agt gtc gag cct gtcagc tgt cct 1152 Gly Asn Lys Val Ser Lys Glu Ser Ser Val Glu Pro Val SerCys Pro 370 375 380 gca gag ccc aaa tct tgt gac aaa act cac aca tgc ccaccg tgc cca 1200 Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro ProCys Pro 385 390 395 400 gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctcttc ccc cca aaa 1248 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu PhePro Pro Lys 405 410 415 ccc aag gac acc ctc atg atc tcc cgg acc cct gaggtc aca tgc gtg 1296 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu ValThr Cys Val 420 425 430 gtg gtg gac gtg agc cac gaa gac cct gag gtc aagttc aac tgg tac 1344 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys PheAsn Trp Tyr 435 440 445 gtg gac ggc gtg gag gtg cat aat gcc aag aca aagccg cgg gag gag 1392 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys ProArg Glu Glu 450 455 460 cag tac aac agc acg tac cgg gtg gtc agc gtc ctcacc gtc tgt cac 1440 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu ThrVal Cys His 465 470 475 480 cag gac tgg ctg aat ggc aag gag tac aag tgcaag gtc tcc aac aaa 1488 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys LysVal Ser Asn Lys 485 490 495 gcc ctc cca gcc ccc atc gag aaa acc atc tccaaa gcc aaa ggg cag 1536 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser LysAla Lys Gly Gln 500 505 510 ccc cga gaa cca cag gtg tac acc ctg ccc ccatcc cgg gat gag ctg 1584 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro SerArg Asp Glu Leu 515 520 525 acc aag aac cag gtc agc ctg acc tgc ctg gtcaaa ggc ttc tat ccc 1632 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val LysGly Phe Tyr Pro 530 535 540 agc gac atc gcc gtg gag tgg gag agc aat gggcag ccg gag aac aac 1680 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly GlnPro Glu Asn Asn 545 550 555 560 tac aag acc acg cct ccc gtg ctg gac tccgac ggc tcc ttc ttc ctc 1728 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser AspGly Ser Phe Phe Leu 565 570 575 tac agc aag ctc acc gtg gac aag agc aggtgg cag cag ggg aac gtc 1776 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg TrpGln Gln Gly Asn Val 580 585 590 ttc tca tgc tcc gtg atg cat gag gct ctgcac aac cac tac acg cag 1824 Phe Ser Cys Ser Val Met His Glu Ala Leu HisAsn His Tyr Thr Gln 595 600 605 aag agc ctc tcc ctg tct ccg ggt aaa tga1854 Lys Ser Leu Ser Leu Ser Pro Gly Lys 610 615 22 617 PRT Homo sapiens22 Met Ser Phe Pro Cys Lys Phe Val Ala Ser Phe Leu Leu Ile Phe Asn 1 510 15 Val Ser Ser Lys Gly Ala Val Ser Lys Glu Ile Thr Asn Ala Leu Glu 2025 30 Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro Ser Phe 3540 45 Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys Thr Ser Asp 5055 60 Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu Thr Phe Lys Glu 6570 75 80 Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile Lys His85 90 95 Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp Thr100 105 110 Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys Ile GlnGlu 115 120 125 Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile Asn ThrThr Leu 130 135 140 Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu AsnLeu Tyr Gln 145 150 155 160 Asp Gly Lys His Leu Lys Leu Ser Gln Arg ValIle Thr His Lys Trp 165 170 175 Thr Thr Ser Leu Ser Ala Lys Phe Lys CysThr Ala Gly Asn Lys Val 180 185 190 Ser Lys Glu Ser Ser Val Glu Asn ValSer Cys Pro Lys Asn Ile Thr 195 200 205 Asn Ala Leu Glu Thr Trp Gly AlaLeu Gly Gln Asp Ile Asn Leu Asp 210 215 220 Ile Pro Ser Phe Gln Met SerAsp Asp Ile Asp Asp Ile Lys Trp Glu 225 230 235 240 Lys Thr Ser Asp LysLys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu 245 250 255 Thr Phe Lys GluLys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu 260 265 270 Lys Ile LysHis Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser 275 280 285 Ile TyrAsp Thr Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu 290 295 300 LysIle Gln Glu Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile 305 310 315320 Asn Thr Thr Leu Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu 325330 335 Asn Leu Tyr Gln Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val Ile340 345 350 Thr His Lys Trp Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys ThrAla 355 360 365 Gly Asn Lys Val Ser Lys Glu Ser Ser Val Glu Pro Val SerCys Pro 370 375 380 Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys ProPro Cys Pro 385 390 395 400 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val PheLeu Phe Pro Pro Lys 405 410 415 Pro Lys Asp Thr Leu Met Ile Ser Arg ThrPro Glu Val Thr Cys Val 420 425 430 Val Val Asp Val Ser His Glu Asp ProGlu Val Lys Phe Asn Trp Tyr 435 440 445 Val Asp Gly Val Glu Val His AsnAla Lys Thr Lys Pro Arg Glu Glu 450 455 460 Gln Tyr Asn Ser Thr Tyr ArgVal Val Ser Val Leu Thr Val Cys His 465 470 475 480 Gln Asp Trp Leu AsnGly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 485 490 495 Ala Leu Pro AlaPro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 500 505 510 Pro Arg GluPro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 515 520 525 Thr LysAsn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 530 535 540 SerAsp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 545 550 555560 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 565570 575 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val580 585 590 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr ThrGln 595 600 605 Lys Ser Leu Ser Leu Ser Pro Gly Lys 610 615 23 1509 DNAHomo sapiens CDS (1)..(1506) mgCTLA4-CTLA4-IgG 23 atg agg acc tgg ccctgc act ctc ctg ttt ttt ctt ctc ttc atc cct 48 Met Arg Thr Trp Pro CysThr Leu Leu Phe Phe Leu Leu Phe Ile Pro 1 5 10 15 gtc ttc tgc aaa gcaatg cac gtg gcc cag cct gct gtg gta ctg gcc 96 Val Phe Cys Lys Ala MetHis Val Ala Gln Pro Ala Val Val Leu Ala 20 25 30 agc agc cga ggc atc gccagc ttt gtg tgt gag tat gca tct cca ggc 144 Ser Ser Arg Gly Ile Ala SerPhe Val Cys Glu Tyr Ala Ser Pro Gly 35 40 45 aaa gcc act gag gtc cgg gtgaca gtg ctt cgg cag gct gac agc cag 192 Lys Ala Thr Glu Val Arg Val ThrVal Leu Arg Gln Ala Asp Ser Gln 50 55 60 gtg act gaa gtc tgt gcg gca acctac atg atg ggg aat gag ttg acc 240 Val Thr Glu Val Cys Ala Ala Thr TyrMet Met Gly Asn Glu Leu Thr 65 70 75 80 ttc cta gat gat tcc atc tgc acgggc acc tcc agt gga aat caa gtg 288 Phe Leu Asp Asp Ser Ile Cys Thr GlyThr Ser Ser Gly Asn Gln Val 85 90 95 aac ctc act atc caa gga ctg agg gccatg gac acg gga ctc tac atc 336 Asn Leu Thr Ile Gln Gly Leu Arg Ala MetAsp Thr Gly Leu Tyr Ile 100 105 110 tgc aag gtg gag ctc atg tac cca ccgcca tac tac ctg ggc ata ggc 384 Cys Lys Val Glu Leu Met Tyr Pro Pro ProTyr Tyr Leu Gly Ile Gly 115 120 125 aac gga acc cag att tat gta aat gataca gaa ccg tgc aat gat tcg 432 Asn Gly Thr Gln Ile Tyr Val Asn Asp ThrGlu Pro Cys Asn Asp Ser 130 135 140 gat aac aat cac acg gcc cag cct gctgtg gta ctg gcc agc agc cga 480 Asp Asn Asn His Thr Ala Gln Pro Ala ValVal Leu Ala Ser Ser Arg 145 150 155 160 ggc atc gcc agc ttt gtg tgt gagtat gca tct cca ggc aaa gcc act 528 Gly Ile Ala Ser Phe Val Cys Glu TyrAla Ser Pro Gly Lys Ala Thr 165 170 175 gag gtc cgg gtg aca gtg ctt cggcag gct gac agc cag gtg act gaa 576 Glu Val Arg Val Thr Val Leu Arg GlnAla Asp Ser Gln Val Thr Glu 180 185 190 gtc tgt gcg gca acc tac atg atgggg aat gag ttg acc ttc cta gat 624 Val Cys Ala Ala Thr Tyr Met Met GlyAsn Glu Leu Thr Phe Leu Asp 195 200 205 gat tcc atc tgc acg ggc acc tccagt gga aat caa gtg aac ctc act 672 Asp Ser Ile Cys Thr Gly Thr Ser SerGly Asn Gln Val Asn Leu Thr 210 215 220 atc caa gga ctg agg gcc atg gacacg gga ctc tac atc tgc aag gtg 720 Ile Gln Gly Leu Arg Ala Met Asp ThrGly Leu Tyr Ile Cys Lys Val 225 230 235 240 gag ctc atg tac cca ccg ccatac tac ctg ggc ata ggc aac gga acc 768 Glu Leu Met Tyr Pro Pro Pro TyrTyr Leu Gly Ile Gly Asn Gly Thr 245 250 255 cag att tat gta att gat ccagaa ccg tgc cca gat tct gca gag ccc 816 Gln Ile Tyr Val Ile Asp Pro GluPro Cys Pro Asp Ser Ala Glu Pro 260 265 270 aaa tct tgt gac aaa act cacaca tgc cca ccg tgc cca gca cct gaa 864 Lys Ser Cys Asp Lys Thr His ThrCys Pro Pro Cys Pro Ala Pro Glu 275 280 285 ctc ctg ggg gga ccg tca gtcttc ctc ttc ccc cca aaa ccc aag gac 912 Leu Leu Gly Gly Pro Ser Val PheLeu Phe Pro Pro Lys Pro Lys Asp 290 295 300 acc ctc atg atc tcc cgg acccct gag gtc aca tgc gtg gtg gtg gac 960 Thr Leu Met Ile Ser Arg Thr ProGlu Val Thr Cys Val Val Val Asp 305 310 315 320 gtg agc cac gaa gac cctgag gtc aag ttc aac tgg tac gtg gac ggc 1008 Val Ser His Glu Asp Pro GluVal Lys Phe Asn Trp Tyr Val Asp Gly 325 330 335 gtg gag gtg cat aat gccaag aca aag ccg cgg gag gag cag tac aac 1056 Val Glu Val His Asn Ala LysThr Lys Pro Arg Glu Glu Gln Tyr Asn 340 345 350 agc acg tac cgg gtg gtcagc gtc ctc acc gtc tgt cac cag gac tgg 1104 Ser Thr Tyr Arg Val Val SerVal Leu Thr Val Cys His Gln Asp Trp 355 360 365 ctg aat ggc aag gag tacaag tgc aag gtc tcc aac aaa gcc ctc cca 1152 Leu Asn Gly Lys Glu Tyr LysCys Lys Val Ser Asn Lys Ala Leu Pro 370 375 380 gcc ccc atc gag aaa accatc tcc aaa gcc aaa ggg cag ccc cga gaa 1200 Ala Pro Ile Glu Lys Thr IleSer Lys Ala Lys Gly Gln Pro Arg Glu 385 390 395 400 cca cag gtg tac accctg ccc cca tcc cgg gat gag ctg acc aag aac 1248 Pro Gln Val Tyr Thr LeuPro Pro Ser Arg Asp Glu Leu Thr Lys Asn 405 410 415 cag gtc agc ctg acctgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1296 Gln Val Ser Leu Thr CysLeu Val Lys Gly Phe Tyr Pro Ser Asp Ile 420 425 430 gcc gtg gag tgg gagagc aat ggg cag ccg gag aac aac tac aag acc 1344 Ala Val Glu Trp Glu SerAsn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 435 440 445 acg cct ccc gtg ctggac tcc gac ggc tcc ttc ttc ctc tac agc aag 1392 Thr Pro Pro Val Leu AspSer Asp Gly Ser Phe Phe Leu Tyr Ser Lys 450 455 460 ctc acc gtg gac aagagc agg tgg cag cag ggg aac gtc ttc tca tgc 1440 Leu Thr Val Asp Lys SerArg Trp Gln Gln Gly Asn Val Phe Ser Cys 465 470 475 480 tcc gtg atg catgag gct ctg cac aac cac tac acg cag aag agc ctc 1488 Ser Val Met His GluAla Leu His Asn His Tyr Thr Gln Lys Ser Leu 485 490 495 tcc ctg tct ccgggt aaa tga 1509 Ser Leu Ser Pro Gly Lys 500 24 502 PRT Homo sapiens 24Met Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro 1 5 1015 Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala 20 2530 Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly 35 4045 Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln 50 5560 Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr 65 7075 80 Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val 8590 95 Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile100 105 110 Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly IleGly 115 120 125 Asn Gly Thr Gln Ile Tyr Val Asn Asp Thr Glu Pro Cys AsnAsp Ser 130 135 140 Asp Asn Asn His Thr Ala Gln Pro Ala Val Val Leu AlaSer Ser Arg 145 150 155 160 Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala SerPro Gly Lys Ala Thr 165 170 175 Glu Val Arg Val Thr Val Leu Arg Gln AlaAsp Ser Gln Val Thr Glu 180 185 190 Val Cys Ala Ala Thr Tyr Met Met GlyAsn Glu Leu Thr Phe Leu Asp 195 200 205 Asp Ser Ile Cys Thr Gly Thr SerSer Gly Asn Gln Val Asn Leu Thr 210 215 220 Ile Gln Gly Leu Arg Ala MetAsp Thr Gly Leu Tyr Ile Cys Lys Val 225 230 235 240 Glu Leu Met Tyr ProPro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr 245 250 255 Gln Ile Tyr ValIle Asp Pro Glu Pro Cys Pro Asp Ser Ala Glu Pro 260 265 270 Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 275 280 285 Leu LeuGly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 290 295 300 ThrLeu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 305 310 315320 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 325330 335 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn340 345 350 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Cys His Gln AspTrp 355 360 365 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys AlaLeu Pro 370 375 380 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly GlnPro Arg Glu 385 390 395 400 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg AspGlu Leu Thr Lys Asn 405 410 415 Gln Val Ser Leu Thr Cys Leu Val Lys GlyPhe Tyr Pro Ser Asp Ile 420 425 430 Ala Val Glu Trp Glu Ser Asn Gly GlnPro Glu Asn Asn Tyr Lys Thr 435 440 445 Thr Pro Pro Val Leu Asp Ser AspGly Ser Phe Phe Leu Tyr Ser Lys 450 455 460 Leu Thr Val Asp Lys Ser ArgTrp Gln Gln Gly Asn Val Phe Ser Cys 465 470 475 480 Ser Val Met His GluAla Leu His Asn His Tyr Thr Gln Lys Ser Leu 485 490 495 Ser Leu Ser ProGly Lys 500 25 33 DNA Artificial Sequence PCR primer, oligonucleotideTNFR1-EDF-EcoRI 25 ccggaattcc ggtctggcat gggcctctcc acc 33 26 37 DNAArtificial Sequence PCR primer, oligonucleotide TNFR1-EDR-IgGh 26cacaagattt gggctctgct gtggtgcctg agtcctc 37 27 37 DNA ArtificialSequence PCR primer, oligonucleotide IgG1-T1F 27 gaggactcag gcaccacagcagagcccaaa tcttgtg 37 28 34 DNA Artificial Sequence PCR primer,oligonucleotide IgG1-R-XbaI 28 gctctagagc tcatttaccc ggagacaggg agag 3429 33 DNA Artificial Sequence PCR primer, oligonucleotideTNFR2-EDF-EcoRI 29 ccggaattcc gggcacccat ggcgcccgtc gcc 33 30 37 DNAArtificial Sequence PCR primer, oligonucleotide TNFR2-EDR-IgGh 30cacaagattt gggctctgcg tcgccagtgc tcccttc 37 31 37 DNA ArtificialSequence PCR primer, oligonucleotide IgG-T2F 31 gaagggagca ctggcgacgcagagcccaaa tcttgtg 37 32 37 DNA Artificial Sequence PCR primer,oligonucleotide TNFR1-CF-BamHI 32 cgcggatccg ggaacatttc actggtccctcacctag 37 33 39 DNA Artificial Sequence PCR primer, oligonucleotideTNFR1-NR-BamHI 33 cgcggatccg tcctcagtgc ccttaacatt ctcaatctg 39 34 36DNA Artificial Sequence PCR primer, oligonucleotide TNFR2-CF-BamHI 34cgcggatcca acgcaactac accctacgcc ccggag 36 35 31 DNA Artificial SequencePCR primer, oligonucleotide TNFR2-NR-BamHI 35 cgcggatccg ctcccttcagctggggggct g 31 36 63 DNA Artificial Sequence PCR primer,oligonucleotide mgTNFR1-TNFR1-IgG-F 36 aaaagcaacg agaccaacaa gacctgcctacacaacgggt ccagggagaa gaacgatagt 60 gtg 63 37 62 DNA Artificial SequencePCR primer, oligonucleotide mgTNFR1-TNFR1-IgG-R 37 ctccctggac ccgttgtgtaggcaggtctt gttggtctcg ttgcttttct tacagttact 60 ac 62 38 45 DNAArtificial Sequence PCR primer, oligonucleotide mgTNFR2-TNFR2-IgG-F 38atggatgcaa actgcacgtc cccggagccc aacagcacat gccgg 45 39 42 DNAArtificial Sequence PCR primer, oligonucleotide mgTNFR2-TNFR2-IgG-R 39gcatgtgctg ttgggctccg gggacgtgca gtttgcatcc at 42 40 36 DNA ArtificialSequence PCR primer, oligonucleotide CD2F-EcoRI 40 ccggaattca tgagctttccatgtaaattt gtagcc 36 41 30 DNA Artificial Sequence PCR primer,oligonucleotide CD2R-PstI 41 ctctgcagga cagctgacag gctcgacact 30 42 25DNA Artificial Sequence PCR primer, oligonucleotide IgG-F-PstI 42atctgcagag cccaaatctt gtgac 25 43 24 DNA Artificial Sequence PCR primer,oligonucleotide CTLA4F-EcoRI 43 ccggaattca tgaggacctg gccc 24 44 30 DNAArtificial Sequence PCR primer, oligonucleotide CTLA4R-PstI 44ctctgcagaa tctgggcacg gttcaggatc 30 45 19 DNA Artificial Sequence PCRprimer, oligonucleotide CD2-NT-F 45 taaagagatt acgaatgcc 19 46 18 DNAArtificial Sequence PCR primer, oligonucleotide CD2-CT-R 46 tgcaggacagctgacagg 18 47 23 DNA Artificial Sequence PCR primer, oligonucleotideCTLA4-NT-F 47 ggataatcat gcacgtggcc cag 23 48 18 DNA Artificial SequencePCR primer, oligonucleotide CTLA4-CT-R 48 tgcagaatct gggcacgg 18 49 43DNA Artificial Sequence PCR primer, oligonucleotide mgCD2-CD2-IgG-F 49cagtgtcgag aatgtcagct gtcctaaaaa tattacgaat gcc 43 50 43 DNA ArtificialSequence PCR primer, oligonucleotide mgCD2-CD2-IgG-R 50 ggcattcgtaatatttttag gacagctgac attctcgaca ctg 43 51 64 DNA Artificial SequencePCR primer, oligonucleotide mgCTLA4-CTLA4-IgG-F 51 atttatgtaa acgatacagaaccgtgcaat gattcggata acaaccacac agcccagcct 60 gctg 64 52 63 DNAArtificial Sequence PCR primer, oligonucleotide mgCTLA4-CTLA4-IgG-R 52aggctgggct gtgtggttgt tatccgaatc attgcacggt tctgtatcgt ttacataaat 60 ctg63

What is claimed is:
 1. A concatameric protein comprising two solubledomains, in which a N-terminus of a soluble domain of a biologicallyactive protein is linked to C- terminus of an identical soluble domainor a different soluble domain of a biologically active protein.
 2. Aconcatameric fusion dimeric protein comprising two monomeric proteinsformed by linkage of a concatamer of two identical soluble extracellulardomains of proteins involving immune response to a hinge region of an Fcfragment of an immunoglobulin molecule, wherein said monomeric proteinsare linked by intermolecular disulfide bonds at the hinge region, andhaving improved stability and therapeutic effects.
 3. The concatamericfusion dimeric protein as set forth in claim 2, wherein theimmunoglobulin molecule is IgG.
 4. The concatameric fusion dimericprotein as set forth in claim 2, wherein the protein involving immuneresponse is selected from the group consisting of cytokines, cytokinereceptors, adhesion molecules, tumor necrosis factor receptors, receptortyrosine kinases, chemokine receptors and other cell surface proteinswhich contain a soluble extracellular domain.
 5. The concatameric fusiondimeric protein as set forth in claim 4, wherein the protein is selectedfrom the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-10, IL-12, IL-17, TNF, TGF, IFN, GM-CSF, G-CSF, EPO, TPO, M-CSF, GHR,IL-13R, IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-9R, IL-15R,TNFR, TGFR, IFNR, interferon-α R, -β R and -γ R, GM-CSFR, G-CSFR, EPOR,cMp1, gp130, Fas (Apo 1), CCR1, CXCR1-4, TrkA, TrkB, TrkC, Htk, REK7,Rse/Tyro-3, hepatocyte growth factor R, platelet-derived growth factorR, Flt-1, CD2, CD4, CD5, CD6, CD22, CD27, CD28, CD30, CD31, CD40, CD44,CD100, CD137, CD150, LAG-3, B7, B61, β-neurexin, CTLA-4, ICOS, ICAM-1,complement R-2 (CD21), IgER, lysosomal membrane gp-1, α2-microglobulinreceptor-related proteins, and sodium-releasing peptide R.
 6. Theconcatameric fusion dimeric protein as set forth in claim 2, wherein themonomeric protein contains an amino acid sequence of SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 18, or SEQ ID NO:
 20. 7. A DNA construct encoding amonomeric protein formed by linkage of a concatamer of two identicalsoluble extracellular domains of a protein involving immune response toa hinge region of an Fc fragment of an immunoglobulin molecule.
 8. TheDNA construct as set forth in claim 7, wherein the immunoglobulinmolecule is IgG.
 9. The DNA construct as set forth in claim 7, whereinthe protein involving immune response is selected from the groupconsisting of cytokines, cytokine receptors, adhesion molecules, tumornecrosis factor receptors, receptor tyrosine kinases, chemokinereceptors and other cell surface proteins which contain a solubleextracellular domain.
 10. The DNA construct as set forth in claim 9,wherein the protein is selected from the group consisting of IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-17, TNF, TGF, IFN,GM-CSF, G-CSF, EPO, TPO, M-CSF, GHR, IL-13R, IL-1R, IL-2R, IL-3R, IL-4R,IL-5R, IL-6R, IL-7R, IL-9R, IL-15R, TNFR, TGFR, IFNR, interferon-α R, -βR and -γ R, GM-CSFR, G-CSFR, EPOR, cMp1, gp130, Fas (Apo 1), CCR1,CXCR1-4, TrkA, TrkB, TrkC, Htk, REK7, Rse/Tyro-3, hepatocyte growthfactor R, platelet-derived growth factor R, Flt-1, CD2, CD4, CD5, CD6,CD22, CD27, CD28, CD30, CD31, CD40, CD44, CD100, CD137, CD150, LAG-3,B7, B61, β-neurexin, CTLA-4, ICOS, ICAM-1, complement R-2 (CD21), IgER,lysosomal membrane gp-1, α2-microglobulin receptor-related proteins, andsodium-releasing peptide R.
 11. The DNA construct as set forth in claim7, wherein the DNA construct contains a nucleotide sequence of SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 17, or SEQ ID NO:
 19. 12. A recombinantexpression plasmid comprising the DNA construct of claim 7 operablylinked thereto.
 13. The recombinant expression plasmid as set forth inclaim 12, wherein the recombinant expression plasmid is a pTR11-Top10′plasmid (accession No.: KCCM 10288), a pTR22-Top10′ plasmid (accessionNo.: KCCM 10289), a pCD22Ig plasmid (accession No.: KCCM 10402), or apCT44Ig plasmid (accession No.: KCCM 10400).
 14. A host cell transformedor transfected with the recombinant expression plasmid of claim
 12. 15.The host cell as set forth in claim 14, wherein the host cell is amammalian cell.
 16. The host cell as set forth in claim 14 or 15,wherein the recombinant expression plasmid is a pTR11-Top10′ plasmid(accession No.: KCCM 10288), a pTR22-Top10′ plasmid (accession No.: KCCM10289), a pCD22Ig plasmid (accession No.: KCCM 10402), or a pCT44Igplasmid (accession No.: KCCM 10400).
 17. The host cell as set forth inclaim 16, wherein the host cell is a TR11Ig-CHO cell line (accessionNo.: KCLRF-BP-00046) or a TR22Ig-CHO cell line (accession No.:KCLRF-BP-00049).
 18. A method of preparing a concatameric fusion dimericprotein in which disulfide bonds are formed between the hinge regions oftwo monomeric proteins, comprising the steps of: culturing thetransformed or transfected host cell of claim 14 under conditionssuitable for expression of a DNA construct encoding a concatamericfusion monomeric protein in which a concatamer of two identical solubleextracellular domains of proteins involving immune response is linked toa hinge region of an Fc fragment of an immunoglobulin molecule; andisolating and purifying a dimeric protein formed by dimerization of theproduced monomeric proteins from culture medium.
 19. The method as setforth in claim 18, wherein the DNA construct encoding a concatamericfusion monomeric protein is prepared by preparing a DNA constructencoding a simple fusion monomeric protein formed by joining a DNAfragment encoding an Fc fragment of an immunoglobulin molecule and a DNAfragment encoding a soluble extracellular domain of a protein involvingimmune response; and joining the prepared DNA construct and a second DNAfragment identical to the DNA fragment encoding a soluble extracellulardomain of a protein involving immune response.
 20. The method as setforth in claim 19, wherein the DNA construct encoding a concatamericfusion monomeric protein contains a glycosylation motif sequence. 21.The method as set forth in claim 20, wherein the glycosylation motifsequence is inserted to a region at which two soluble extracellulardomains are joined.
 22. The method as set forth in claim 19, wherein theconcatameric fusion monomeric protein contains a leader sequence. 23.The method as set forth in claim 22, wherein the concatameric fusionmonomeric protein is CTLA-4, and the leader sequence has an amino acidsequence of MACLGFQRHKAQKNLAARTWPCTLLFFIPVFCKA.
 24. The method as setforth in claim 23, wherein the leader sequence has an amino acidsequence of MRTWPCTLLFFIPVFCKA excluding ACLGFQRHKAQKNLAA.
 25. Themethod as set forth in any of claims 18 to 24, wherein the host cell isa mammalian cell.
 26. A concatameric fusion dimeric protein comprisingtwo monomeric proteins formed by linkage of a concatamer of twoidentical soluble extracellular domains of proteins involving immuneresponse to the hinge region of Fc fragment of an immunoglobulinmolecule, wherein said monomeric proteins are linked by formation ofintermolecular disulfide bonds at the hinge region and glycosylated, andhaving improved stability and therapeutic effects.
 27. The concatamericfusion dimeric protein as set forth in claim 26, wherein the monomericprotein contains an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 22, or SEQ ID NO:
 24. 28. A DNA construct encoding amonomeric protein formed by linkage of a concatamer of two identicalsoluble extracellular domains of proteins involving immune response to ahinge region of an Fc fragment of an immunoglobulin molecule andcontaining glycosylation motif peptides.
 29. The DNA construct as setforth in claim 28, wherein the DNA construct contains an amino acidsequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 21, or SEQ ID NO:23.
 30. A recombinant expression plasmid operably linked to the DNAconstruct of claim
 28. 31. The recombinant expression plasmid as setforth in claim 30, wherein the recombinant expression plasmid is apTR11Ig-MG plasmid (accession No.: KCCM 10404), a pTR22Ig-MG plasmid(accession No.: KCCM 10407), a pCD22Ig-MG plasmid (accession No.: KCCM10401), or a pCT44Ig-MG plasmid (accession No.: KCCM 10399).
 32. A hostcell transformed or transfected with the recombinant expression plasmidof claim
 30. 33. The host cell as set forth in claim 32, wherein thehost cell is a mammalian cell.
 34. A pharmaceutical or diagnosticcomposition comprising the dimeric protein of claim
 2. 35. Apharmaceutical or diagnostic composition comprising the glycosylateddimeric protein of claim 26.